Thiazole compounds, methods for preparation and use thereof

ABSTRACT

The present invention relates to thiazole compounds of formula I, the method for preparation and use thereof. More specifically, the present invention relates to novel derivatives of natural product largazole, the method for preparing them and their uses for treatments against tumor and multiple sclerosis as inhibitors of histone deacetylase.

FIELD OF TECHNOLOGY

The present invention relates to a class of compounds of thiazoles, themethod for their preparation and use thereof. More specifically, thepresent invention relates to novel derivatives of natural productlargazole, the method for preparing them and their uses for treatmentsagainst tumor and multiple sclerosis as inhibitors of histonedeacetylase (HDAC).

BACKGROUND ART

Tumor is the second largest disease after the cardiovascular diseaseworldwide and its incidence rate is still rising in recent years. Thetreatment of cancer has always been a problem troubling human beings.Because of the lack of selectivity of targets, traditional chemotherapydrugs tend to produce more serious toxic side effect. This situationrequires people to develop antitumor drugs of specific molecular targetswith high efficiency and low toxicity. It has become an importantdirection of research and development of antitumor drugs nowadays to usea key enzyme of cell signal transduction pathway related to tumor celldifferentiation, proliferation and metastasis as a target of drugscreening, and find new anticancer medicine selectively acting on thisspecific target with high efficiency, low toxicity and high specificity.Histone deacetylase (HDAC) is such a key enzyme.

Multiple Sclerosis (MS) is a kind of typical autoimmune diseases. Thepathology of MS disease includes acute inflammation of the centralnervous system and demyelination, which is one of the most importantincentives leading to noninvasive paralysis of the nervous system anddisability among young people. The clinical manifestation of MS isheterogeneous. More than 80% of the patients are manifested as relievedrelapse phenotype. Since pathogenesis of the disease is unknown andsensitive diagnostic signs are still lacking at present, the diagnosisof MS still only depends on multiple characteristic of the disease intime and space. In addition, a lot of other diseases such asophthalmoneuromyelitis have symptoms extremely similar to MS. Thereforethe MS treatment even the diagnosis remains very difficult at present.

The current findings indicate that CD4⁺ T cells play an important rolein the pathogenesis of MS, at least initiating MS at an early stage.Previous studies suggest that T_(H)1 cell (characterized in producingIFN-γ) plays an important role in the occurrence of the diseases. Withthe discovery of T_(H)17, more and more evidence shows that the functionof latter in the pathogenesis of MS is no inferior than that of T_(H)1,for example, the mice with less quantity of T_(H)17 cells were notsusceptible to EAE (Experimental Autoimmune Encephalomyelitis), T_(H)17cells were identified in the brain tissue of the MS patients and etc.

Chromatin histone acetylation and deacetylation is one of the criticalprocesses regulating gene expression, while abnormal gene expression isthe molecular biological basis of the occurrence of tumor and somegenetic and metabolic diseases. Degree of acetylation of histones iscoordinately controlled by histone acetyltransferase (HAT) and histonedeacetylase (HDAC). When HDAC is over expressed and then recruited bytranscription factor, it will lead to the abnormal inhibition ofspecific genes, thus resulting in the occurrence of tumors and otherdiseases.

HDACs have eighteen subtypes, which can be divided into four classes,namely class I (HDAC 1, 2, 3, 8), class II (HDAC 4, 5, 6, 7, 9), classIII (SIRT 1-7) and class IV (HDAC 11) respectively. (Johnstone, R. W.2002 Nature Rev. Drug Disc. 1:287). It is reported that activity of HDACis relevant to the occurrence of cancer (Archer, S. Y. etc. 1998 Proc.Nat. Acad. Sci. USA, 95: 6791-6796). When HDAC is over expressed, itwill inhibit in vivo gene expression of natural tumor-inhibiting factorssuch as p53 (Gu, W. etc. 1997 Cell, 90: 595-606). However, inhibitors ofHDAC (HDACi) enhance chromatin histone acetylation level, thus resultingin activation of expression of specific genes, accordingly resulting indifferentiation of cells or apoptosis of cancer cells. Clinical studiesshow that the high level of acetylation of histones can be achieved byinhibiting the activity of HDAC.

The HDAC is that have already been discovered at present can be dividedinto the following categories on the basis of structure: short-chainfatty acids; hydroxamic acids; electrophilic epoxy-ketons group;o-phenylenediamines and macrocyclic peptides (Miller, T. A. etc. 2003 J.Med. Chem. 46:5097; Rosato, R. R. etc. 2004 Expert. Opin. Invest. Drugs13:21; Monneret, C., 2005 Eur. J. Med. 40:1; Yoo, C. B. etc. 2006 Nat.Rev. Drug Discovery 5:37). The HDACi structure-activity relationshipstudies have shown that most HDAC is can be segmented into surfacerecognition structural domain, zinc chelating region (ZBG) andhydrophobic aliphatic chain connecting the former two parts (Marks, P.2007 Oncogene 26:1351).

Histone deacetylase (HDAC) is an important protein for epigeneticregulation, which regulates chromatin remodeling, gene expression andfunctions of various proteins including transcription factors, histones,cytoskeletal proteins and etc. HDAC is are a class of small moleculecompounds that can block the activity of HDAC and lead to cell cyclearrest, cell differentiation and apoptosis, which is the reason why theyhave been applied in tumor therapy. Recent experimental results showthat HDAC is may also have anti-inflammatory and immunemodulatoryeffects. Camelo et al. found that the in the mice model of MS(Experimental autoimmune encephalomyelitis, EAE) HDACi TSA caneffectively inhibit T cell invasion to the central nervous system ofmice, thereby reducing the clinical symptoms of the disease. Chung etal. found that, the HDAC is phenyl butyric acid and TSA can inhibitexpression of TNF-alpha in animal arthritis models and reduce theinfiltration of mononuclear cells, thereby reducing the symptoms of thedisease. Studies carried out by Bosisio et al. proved that HDACi caninhibit the antigen-presenting dendritic cell from secreting cytokinesin favor of producing T_(H)1 and T_(H)17, which includes IL-12, IL-23etc. Studies carried out by Tao et al. showed that HDACi can enhance thedifferentiation of the suppressive Treg cell and inhibitory function onT_(H)1 and T_(H)17 cells. The above-mentioned studies showed that theinhibition of HDAC is closely related to the occurrence and developmentof autoimmune diseases. HDACi with lower toxicity, better selectivitywill contribute to the treatment of autoimmune diseases, especially tothe treatment of MS diseases.

Largazole is a highly functionalized sixteen-membered cyclic peptidelactone isolated from the Florida marine cyanobacteria Symploa sp.(Taori, K., et al. 2008 J. Am. Chem. Soc. 130:1806-1807; Ying, Y. et al.2008 J. Am. Chem. Soc. 130:8455-8459), and its structure is shown asfollows. Largazole has a novel skeleton structure, which includes: thetandem motif of dihydro-thiazole ring substituted by 4-methyl andthiazole ring, L-valine and (3S,4E)-3-hydroxy-7-sulfydryl-4-heptenoicacid. Pharmacological experiments show that largazole is able toselectively inhibit the growth of breast cancer cells (MDA-MB-231) andfibroblast osteosarcoma cells (U2OS), while showing less effect onnormal mammary epithelial cells (NMuMG) and normal fibroblasts (NIH3T3).Later studies showed that largazole is able to selectively inhibit classI HDAC. Based on this knowledge of largazole, the inventors optimizedand modified the structure of largazole, and evaluated their activities,thereby obtaining a series of compounds with potential for furtherdevelopment.

SUMMARY OF THE INVENTION

One object of the present invention is to design and synthesize a novelclass of thiazole compounds, which can be used as histone deacetylaseinhibitors, thus providing new ways for discovery of new drugs againsttumor and multiple sclerosis.

Another object of the present invention is to provide methods forpreparing of said thiazole compounds.

Another object of the present invention is to provide the use of saidthiazole compounds.

The thiazole compounds of present invention have the structure ofgeneral formula I:

wherein,

R₁ is R_(1a), R_(1b) or R_(1c):

In which,

R_(4a) and R_(5a) each independently are A group or C₁-C₆ alkyloptionally substituted by tert-butoxycarbonylamino;

R_(4b) and R_(5b) each independently are A group; or,

R_(4b) and R_(5b) together with the carbon atom to which they areattached form a 3 to 10-membered cyclic hydrocarbon or a 3 to10-membered heterocycle containing 1 to 3 heteroatoms selected from N, Oand S;

R_(4c), R_(5c) and R₆ each independently are A group;

The said A group is hydrogen, halogen, hydroxyl, nitro, C₃-C₆cycloalkyl, C₁-C₆ alkoxyl, C₁-C₆ alkoxyl optionally substituted byC₆-C₁₀ aryl, amino, C₁-C₆ alkylamino, C₁-C₆ alkylamino optionallysubstituted by C₆-C₁₀ aryl, C₁-C₆ alkyl, C₁-C₆ alkyl optionallysubstituted by hydroxyl, C₁-C₆ alkyl optionally substituted by C₁-C₄alkoxyl, C₁-C₆ alkyl optionally substituted by fluoro, C₁-C₆ alkyloptionally substituted by C₆-C₁₀ aryl, C₂-C₆ alkenyl, C₂-C₆ alkenyloptionally substituted by hydroxyl, C₂-C₆ alkenyl optionally substitutedby C₁-C₄ alkoxyl, C₂-C₆ alkenyl optionally substituted by fluoro, C₂-C₆alkenyl group optionally substituted by C₆-C₁₀ aryl, C₂-C₆ alkynyl,C₂-C₆ alkynyl optionally substituted by hydroxyl, C₂-C₆ alkynyloptionally substituted by C₁-C₄ alkoxyl, C₂-C₆ alkynyl optionallysubstituted by fluoro, C₂-C₆ alkynyl optionally substituted by C₆-C₁₀aryl, C₆-C₁₀ aryl, or 5 to 7-membered aromatic heterocycle containing1-3 heteroatoms selected from N, O and S;

R₂ is hydrogen, halogen, hydroxyl, C₁-C₆ alkoxyl, C₁-C₈ alkoxyloptionally substituted by C₆-C₁₀ aryl, amino, C₁-C₆ alkylamino, C₁-C₆alkylamino optionally substituted by C₆-C₁₀ aryl, C₁-C₆ alkyl, C₃-C₆cycloalkyl, C₃-C₆ cycloalkyl optionally substituted by C₁-C₆ alkyl,C₁-C₆ alkyl optionally substituted by one or more substituent groupsindependently selected from hydroxyl, C₁-C₄ alkoxyl, halogen, benzyloxyland C₆-C₁₀ aryl, C₂-C₈ alkenyl, C₂-C₆ alkenyl group optionallysubstituted by one or more substituent groups independently selectedfrom hydroxyl, C₁-C₄ alkoxyl, halogen and C₆-C₁₀ aryl, C₂-C₆ alkynyl,C₂-C₈ alkynyl optionally substituted by one or more substituent groupsindependently selected from hydroxyl, C₁-C₄ alkoxyl, halogen and C₆-C₁₀aryl, C₆-C₁₀ aryl, C₆-C₁₀ aryl optionally substituted by halogen ornitro, or 5-7 membered aromatic heterocycle containing 1 to 3heteroatoms selected from N, O and S;

n is 0, 1 or 2;

X is —N(R₇)— or

wherein R₇ is hydrogen or C₁-C₆ alkyl;

Y may do not exist or exist as —(C₁-C₁₀ alkyl)-, —(C₂-C₉ alkenyl)-,—(C₆-C₁₀ aryl)-, —(C₁-C₆ alkyl)-(C₆-C₁₀ aryl)-, —(C₆-C₁₀ aryl)-(C₂-C₆alkenyl)-, —(C₃-C₆ cycloalkyl)-, —(C₁-C₅ alkyl)-C(O)—NH—(C₁-C₅ alkyl)-,—(C₁-C₅ alkyl)-C(O)—O—(C₁-C₅ alkyl)- or —(C₁-C₅ alkyl)-C(O)—O—(C₂-C₉alkenyl)-;

R₃ is R_(3a), R_(3b), R_(3c), R_(3d) or R_(3e):

R₈ is hydrogen or C₁-C₆ alkylcarbonyl; Preferably, R₈ is hydrogen;

R₉ is hydrogen or C₁-C₁₀ alkylcarbonyl.

In a further embodiment of present invention, wherein,

In the general formula I:

wherein:

R₁ is R_(1a);

n=0;

R₂, X, Y and R₃ are defined as above.

specifically as the following general formula II_(a):

In the general formula II_(a):

R_(4a) and R_(5a) are defined as above; Preferably, R_(4a) and R_(5a)each independently are hydrogen, C₁-C₆ alkyl or C₁-C₆ alkyl optionallysubstituted by tert-butoxycarbonylamino.

In another further embodiment of present invention, wherein,

In the general formula I:

wherein:

R₁ is R_(1b);

n=0;

R₂, X, Y and R₃ are defined as above, specifically as the followinggeneral formula II_(b):

In the formula II_(b):

R_(4b) and R_(5b) are defined as above; Preferably, R_(4b) and R_(5b)each independently are hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl or C6-C10aryl; or, R_(4b) and R_(5b) together with the carbon atom to which theyare attached form a 3 to 10-membered cyclic hydrocarbon.

In another further embodiment of present invention, wherein,

In the general formula I:

wherein

R₁ is R_(1c);

n=0;

R₂, X, Y and R₃ are defined as above,

specifically as the following general formula II_(c):

In the formula II_(c):

R_(4c), R_(5c) and R₆ are defined as above;

Preferably, R_(4c), R_(5c) and R₆ each independently are hydrogen orC₁-C₆ alkyl.

In a more preferable embodiment of present invention, said X is —NH, n=0and R₂, R₃, R_(4b), R_(5b) and Y are defined as above, the compound ofpresent invention has the following structure of general formula III:

In a particularly preferable embodiment of present invention, said X is—NH, and n=0, while R₃ is R_(3a), the compound of present invention hasthe following structure of general formula IV:

Wherein, R₂, R₃, R_(4b), R_(5b) and Y are defined as above.

R₂ preferably is hydrogen, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, C₁-C₆ alkyloptionally substituted by hydroxyl, C₃-C₆ cycloalkyl optionallysubstituted by C₁-C₆ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkenyl optionallysubstituted by hydroxyl, C₆-C₁₀ aryl, C₁-C₆ alkyl optionally substitutedby C₆-C₁₀ aryl or benzyloxyl, C₂-C₈ alkenyl optionally substituted byC₆-C₁₀ aryl, C₂-C₈ alkoxyl optional substituted by C₆-C₁₀ aryl, C₆-C₁₀aryl optionally substituted by halogen, or C₆-C₁₀ aryl optionallysubstituted by nitro;

Preferably, R_(4b) and R_(5b) each independently are hydrogen, fluoro,C₁-C₆ alkyl, C₁-C₆ alkyl optionally substituted by hydroxyl, C₁-C₆ alkyloptionally substituted by fluoro, C₃-C₆ cycloalkyl, C₂-C₆ alkenyl, C₂-C₆alkenyl optionally substituted by hydroxyl, C₂-C₆ alkenyl optionallysubstituted by fluoro, or C₆-C₁₀ aryl; or, R_(4b) and R_(5b) togetherwith the carbon atom to which they are attached form a 3 to 10-memberedcyclic hydrocarbon or a 3 to 10-membered heterocycle containing 1 to 3heteroatoms selected from N, O and S;

Y preferably is —(C₁-C₈ alkyl)-, —(C₆-C₁₀ aryl)-, —(C₁-C₆ alkyl)-(C₆-C₁₀aryl), or —(C₆-C₁₀ aryl)-(C₂-C₆ alkenyl)-.

The thiazole compounds of general formula I of present invention,specifically are as follows:

Moreover present invention provides methods for preparing the thiazolecompound of general formula I, wherein, the method can be employed byone of the following Route one to five;

In Route one:

R₁, R₂, n, X and Y each have the same meaning as above; Specifically,compound 1 reacts with active zinc powder in THF to form zinc reagentcompound 2; Negishi coupling reaction of compound 2 with2-bromo-thiazole compound catalyzed by palladium acetate andtriphenylphosphine in toluene affords compound 4. Hydrolysis of compound4 in sodium hydroxide/methanol-water produces compound 5 which is thecorresponding acid of compound 4; Condensation reaction of compound 5with various nucleophilic reagents of the formula HX—Y—COOMe isconducted using condensing agents such as EDCI, HOBt and i-Pr₂NEt in DMFto obtain compound 6; Compound 6 reacts with the freshly-made methanolsolution of hydroxy amine to produce said compound I_(a);

In Route two:

R₁, R₂, R₈, n, X and Y each have the same meaning as above;

Specifically, compound 6 is hydrolyzed by alkali in methanol orTHF/water to obtain Compound I_(e); Condensation reaction of compoundI_(e) with mono-Boc-protected o-phenylenediamine is conducted usingcondensing agents such as EDCI, HOBt and i-Pr₂NEt in DMF to obtaincompound 7; Boc protecting group of compound 7 is removed inhydrochloric acid ethyl acetate solution to obtain said compounds I_(b),or compound 7 reacts with various nucleophile of formula R₈H to obtainsaid compound I_(b);

In Route three:

R₁, R₂, n, X and Y each have the same meaning as above;

Specifically, condensation reaction of compound 5 with variousnucleophile of the formula HX—Y—NHCOCH₂STrt is conducted usingcondensing agents such as EDCI and DMAP as base in dichloromethane toobtain compound 8; Boc protecting group of compound 8 is removed by thetrifluoroacetic acid to obtain said compound I_(e);

In Route four:

R₁, R₂, R₉, n, X and Y each have the same meaning as above;

Specifically, condensation reaction of compound 5 with variousnucleophile of the formula HX—Y—SR₉ is conducted using condensing agentssuch as EDCI, HOBt and i-Pr₂NEt in DMF to obtain said compound I_(d);

In Route five:

R₂, R₃, R_(4c), R_(5c), R₆, n, X and Y each have the same meaning asabove;

Specifically, reaction of compound 9 with Lawesson's agent affordscompound 10; Hantzsch reaction of compound 10 with ethyl bromopyruvatesubstituted by R₂ produces bisthiazole compound 11; Compound 11 ishydrolyzed by alkaline in methanol/water to afford compound 12;Condensation reaction of compound 12 with various nucleophile of theformula HX—Y—R₃ is conducted using condensing agents such as EDCI, HOBtand i-Pr₂NEt in DMF to obtain said compound II_(e).

The thiazole compound of general formula I of present invention can beused for preparing the pharmaceuticals as histone deacetylaseinhibitors, therefore, it can be used for preparing the pharmaceuticalsagainst tumor and multiple sclerosis. The cell lines of said tumorinclude colon cancer cell HCT-116, pancreatic cancer cell Bx-PC3,leukemia cell HL60, human lung adenocarcinoma cell A549, breast cancercell MDA-MB-231, and mammary epithelial cell HMEC. Thus, it can be usedfor the treatment of colon cancer, pancreatic cancer, leukemia, lungcancer or breast cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the clinical score of EAE which was effectively alleviated byHDACi CFH367-C.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention will hereinafter be further illustrated by thefollowing examples, but not limited to such examples.

EXAMPLES FOR PREPARING COMPOUNDS

In the following embodiments of preparation, NMR will be determined byMercury-Vx 300M instrument made by Varian, NMR calibration: δ H 7.26 ppm(CDCl₃), 2.50 ppm (DMSO-d₆), 3.15 ppm (CD₃OD); The reagents are mainlyprovided by Shanghai Chemical Reagent Company; Silica gel plates for TLCthin layer chromatography are produced by Shandong Yantai Huiyou silicagel Development Co. Ltd., Type HSGF 254; Silica gel for normal phasecolumn chromatography used in purification of the compounds are producedby a branch of Shandong Tsingtao Marine Chemical Factory, Type zcx-11,200-300 meshes.

Example 1 of Preparation (Compound Number CFH367-C)

The active Zn powder (298 mg, 4.58 mmol) was dissolved in redistilledanhydrous THF (20 mL). The inner air was replaced by N₂. Compound 13(875 mg, 5.34 mmol) was added dropwise, while the dropping speed wascontrolled to prevent from vigorous boiling. After such addition, thereaction mixture was refluxed for 1.5 h, and then was naturally cooleddown to obtain zinc reagent 14.

Compound 15 (1 g, 3.82 mmol) was dissolved in anhydrous toluene (20 mL).The inner air was replaced by N₂. The above-described Zinc reagent 14 isintroduced into the reaction mixture, then the catalyst palladiumacetate (43 mg, 0.19 mmol) and triphenylphosphine (100 mg, 0.38 mmol)were added. The reaction mixture was heated to 80-90° C. for a whileuntil the crude material compound 15 was all consumed. After removal ofinsoluble substance by filtration, the organic phase was concentratedand then diluted with CH₂Cl₂(50 mL). After adding 1N HCl (30 mL) withstirring for 10 min, the organic phase was separated. The aqueous phasewas extracted by CH₂Cl₂ (30 mL) twice. The combination of all extractedorganic phases was concentrated and then subjected to silica gel columnflash chromotography (PE/EtOAc=4:1) to obtain compound 16 (750 g, 73.5%,yellow grease), ¹H NMR (300 MHz, CDCl₃) δ 7.85 (d, J=3.0 Hz, 1H), 7.45(d, J=3.0 Hz, 1H), 3.96 (s, 3H), 3.10-3.14 (m, 1H), 1.31-1.38 (m, 2H),0.84-0.89 (m, 2H).

Compound 16 (700 mg, 2.63 mmol) was dissolved in MeOH/H₂O (30/6 mL), andthen solid NaOH (210 mg, 5.26 mmol) was added in. The reaction mixturewas heated to reflux for 1 h. After completion of reaction, the obtainedmixture was concentrated under reduced pressure to remove most of MeOHand then diluted with H₂O. The aqueous phase was then acidified to pH=2by 1N hydrochloric acid, extracted by EtOAc (20 mL×3 times), and washedwith brine (30 mL). The organic phase was dried by anhydrous Na₂SO₄ andthen concentrated to obtain a solid compound 17 (660 mg, 99.5%, paleyellow solid) which can be used directly in the next reaction.

Compound 17 (100 mg, 0.40 mmol) was dissolved in dry DMF (5 mL), thencooled to 0° C. in an ice-bath. EDCI (76 mg, 0.40 mmol) and HOBt (54 mg,0.40 mmol) were added and stirred for 10 min at 0° C. Compound 18 (73mg, 0.44 mmol) and i-Pr₂NEt (120 mg, 1.20 mmol) were added and stirredfor 12 h at room temperature. The obtained mixture was diluted with 20mL water, and then extracted by EtOAc (15 mL×3 times). The combinedorganic phases was washed by 1N hydrochloric acid (15 mL), saturatedsodium bicarbonate solution (15 ml) and brine (20 mL) in sequence, andthen dried by anhydrous Na₂SO₄. After removal of solvent by evaporatingunder a reduced pressure, the obtained residue was purified by silicagel column chromatography (PE/EtOAc=2:1) to obtain product compound 19(104 mg, 71.7%, pale yellow grease). ¹H NMR (300 MHz, CDCl₃) δ7.74 (d,J=3.0 Hz 1H), 7.42 (t, J=5.4 Hz, 1H), 7.36 (d, J=3.0 Hz, 1H), 3.58 (s,3H), 3.37 (q, J=6.3 Hz, 2H), 2.30 (t, J=6.6 Hz, 2H), 1.58-1.69 (m, 4H),1.19-1.25 (m, 2H), 0.68-0.74 (m, 2H).

Hydroxylamine hydrochloride (197 mg, 2.8 mmol) was suspended in MeOH (15mL), and then KOH (235 mg, 4.20 mmol) was added in and stirred for 5min. The insoluble substance was removed by filtration and the filtratewas collected for use. After compound 19 (104 mg, 0.28 mmol) wasdissolved in anhydrous MeOH (10 ml), freshly-prepared above-mentionedMeOH solution of hydroxylamine hydrochloride was added in and stirredfor 1 h at room temperature. Reaction was monitored by TLC. The obtainedsolution was diluted with EtOAc, then neutralized to pH=5-6 by 1Nhydrochloric acid, After removal of solvent by evaporating under reducedpressure, the obtained mixture was sequentially extracted by EtOAc (15mL×3 times), and washed by brine (10 mL), dried by anhydrous Na₂SO₄ andconcentrated to obtain a crude product which was purified by columnchromatography (CHCl₃/MeOH=20:1-10:1) to obtain product CFH367-C (71 mg,68.3%, pale yellow solid). ¹H NMR (300 MHz, CD₃OD) δ 8.36 (t, J=5.4 Hz,1H), 7.88 (d, J=3.0 Hz 1H), 7.74 (d, J=3.0 Hz 1H), 3.41 (q, J=6.6 Hz,2H), 3.31-3.36 (m, 1H), 2.16 (t, J=6.6 Hz, 2H), 1.64-1.69 (m, 4H),1.29-1.34 (m, 2H), 0.82-0.86 (m, 2H).

The following compounds could be synthesized using the same method asabove:

Compound Structural formula ¹H NMR (CD₃OD, 300 MHz) data CFH326

δ 8.53 (t, J = 5.4 Hz, 1H), 8.27 (s, 1H), 7.92 (d, J = 3.0 Hz, 1H), 7.89(s, 1H), 7.77 (d, J = 3.0 Hz 1H), 3.43 (q, J = 6.0 Hz, 2H), 2.16 (t, J =6.6 Hz, 2H), 1.66-1.69 (m, 4H) CFH340-M

δ 8.53 (t, J = 5.4 Hz, 1H), 8.27 (s, 1H), 7.92 (d, J = 3.0 Hz, 1H), 7.89(s, 1H), 7.77 (d, J = 3.0 Hz, 1H), 3.43 (q, J = 6.0 Hz, 2H), 2.16 (t, J= 6.6 Hz, 2H), 1.66-1.69 (m, 4H) CFH355

δ 8.33 (t, J = 5.4 Hz, 1H), 7.89 (d, J = 3.3 Hz, 1H), 7.73 (d, J = 3.3Hz, 1H), 3.37 (q, J = 6.6 Hz, 2H), 3.31 (q, J = 6.9 Hz, 2H), 2.16 (t, J= 6.3 Hz, 2H), 1.64-1.67 (m, 4H), 1.35 (t, J = 6.9 Hz, 3H) CFH369

δ 8.35 (t, J = 5.4 Hz, 1H), 7.90 (d, J = 3.3 Hz, 1H), 7.74 (d, J = 3.3Hz, 1H), 4.32- 4.36 (m, 1H), 3.41 (q, J = 6.3 Hz, 2H), 2.16 (t, J = 6.3Hz, 2H), 1.67-1.69 (m, 4H), 1.37 (d, J = 6.9 Hz, 6H) CFH383

δ 8.38 (t, J = 5.4 Hz, 1H), 7.92 (d, J = 3.3 Hz, 1H), 7.76 (d, J = 3.3Hz, 1H), 3.40 (q, J = 6.0 Hz, 2H), 3.34 (t, J = 6.0 Hz, 2H), 2.19 (t, J= 6.6 Hz, 2H), 1.72-1.79 (m, 6H), 1.41-1.54 (m, 2H), 1.01 (t, J = 7.5Hz, 3H) CFH382

(DMSO-d₆) δ 10.36 (s, 1H), 8.68 (s, 1H), 8.36 (t, J = 5.7 Hz, 1H),7.99(d, J = 3.0 Hz, 1H), 7.96(d, J = 3.0 Hz, 1H), 3.25 (q, J = 5.7 Hz,2H), 3.20 (d, J = 7.2 Hz, 2H), 1.96 (t, J = 5.4 Hz, 2H), 1.90-1.92 (m,1H), 1.51 (m, 4H), 0.91 (d, J = 6.6 Hz, 6H) CFH340

(DMSO-d₆) δ 10.61 (s, 1H), 8.84 (s, 1H), 8.46 (t, J = 6.9 Hz, 1H), 8.0(d, J = 2.7 Hz, 2H), 3.81 (d, J = 5.4 Hz, 2H), 3.21 (d, J = 6.9 Hz, 2H),1.92-1.96 (m, 1H), 0.93 (d, J = 6.6 Hz, 6H) CFH354

(DMSO-d₆) δ 10.52 (s, 1H), 8.41 (t, J = 5.7 Hz, 1H), 7.97 (d, J = 2.4Hz, 2H), 3.48 (q, J = 5.7 Hz, 2H), 3.20 (d, J = 6.0 Hz, 2H), 2.13 (t, J= 5.4 Hz, 2H), 1.90-1.92 (m, 1H), 0.91 (d, J = 6.0 Hz, 6H) CFH368

(DMSO-d₆) δ 10.42 (s, 1H), 8.73 (s, 1H), 8.41 (t, J = 5.7 Hz, 1H), 7.97(d, J = 3.0 Hz, 1H), 7.95 (d, J = 3.0 Hz, 1H), 3.27 (q, J = 6.6 Hz, 2H),3.19 (d, J = 7.2 Hz, 2H), 2.02 (t, J = 7.2 Hz, 2H), 1.89-1.93 (m, 1H),1.74-1.86 (m, 2H), 0.90 (d, J = 6.6 Hz, 6H) CFH396

(DMSO-d₆) δ 10.39 (s, 1H), 8.33 (t, J = 5.4 Hz, 1H), 7.97 (d, J = 3.3Hz, 1H), 7.93 (d, J = 3.3 Hz, 1H), 3.24 (q, J = 6.3 Hz, 2H), 3.19 (d, J= 7.2 Hz, 2H), 1.95 (t, J = 7.5 Hz, 2H), 1.87-1.91 (m, 1H), 1.49-1.54(m, 4H), 1.26-1.28 (m, 2H), 0.91 (d, J = 6.3 Hz, 6H) CFH410

δ 8.26 (d, J = 3.3 Hz, 1H), 7.85 (d, J = 6.6 Hz, 1H), 7.67 (d, J = 3.3Hz, 1H), 3.30 (q, J = 7.2 Hz, 2H), 3.16 (d, J = 7.2 Hz, 2H), 2.07 (t, J= 7.5 Hz, 2H), 1.89-1.97 (m, 1H), 1.54-1.57 (m, 4H), 1.25-1.33 (m, 4H),0.92 (d, J = 6.6 Hz, 6H) CFH424

(DMSO-d₆) δ 10.33 (s, 1H), 8.66 (s, 1H), 8.34 (t, J = 5.7 Hz, 1H), 7.99(d, J = 3.3 Hz, 1H), 7.97 (d, J = 3.0 Hz, 1H), 3.23-3.25 (m, 2H), 3.20(d, J = 7.2 Hz, 2H), 1.96 (t, J = 7.2 Hz, 2H), 1.86-1.91 (m, 1H),1.48-1.52 (m, 4H), 1.19-1.26 (m, 6H), 0.92 (d, J = 6.6 Hz, 6H) CFH381

δ 8.44 (t, J = 5.4 Hz, 1H), 7.92 (d, J = 3.3 Hz, 1H), 7.81-7.89 (m, 1H),7.78 (d, J = 3.3 Hz, 1H), 5.81 (d, J = 17.7 Hz, 1H), 5.51 (d, J = 10.8Hz, 1H), 3.40 (q, J = 5.7 Hz, 2H), 2.15 (t, J = 7.5 Hz, 2H), 1.63-1.68(m, 4H) CFH395

δ 8.33 (t, J = 5.4 Hz, 1H), 7.89 (s, 2H), 7.73 (s, 1H), 7.54 (d, J =15.9 Hz, 1H), 6.26-6.34 (m, 1H), 3.39 (q, J = 5.7 Hz, 2H), 2.48-2.50 (m,1H), 2.16 (t, J = 6.6 Hz, 2H), 1.63-1.68 (m, 4H), 1.11 (d, J = 6.6 Hz,6H) CFH367

(DMSO-d₆) δ 10.37 (s, 1H), 8.69 (s, 1H), 8.42 (t, J = 5.4 Hz, 1H), 8.00(s, 2H), 7.58 (d, J = 16.2 Hz, 1H), 6.35 (m, 1H), 3.27 (m, 2H), 1.98 (t,J = 6.9 Hz, 2H), 1.73 (d, J = 6.6 Hz, 3H), 1.51 (m, 4H) CFH352

(DMSO-d₆) δ 10.36 (s, 1H), 8.69 (s, 1H), 8.52 (t, J = 5.4 Hz, 1H), 8.04(s, 2H), 7.83 (dd, J = 17.7, 11.1 Hz, 1H), 5.83 (d, J = 17.7 Hz, 1H),5.55 (d, J = 11.1 Hz, 1H), 3.28 (m, 2H), 1.98 (t, J = 6.9 Hz, 2H), 1.51(m, 4H) CFH399

δ 8.47 (t, J = 5.4 Hz, 1H), 7.91 (d, J = 3.0 Hz, 1H), 7.74 (d, J = 3.0Hz, 1H), 3.58 (s, 2H), 3.41 (q, J = 6.0 Hz, 2H), 2.16 (t, J = 6.3 Hz,2H), 1.63-1.67(m, 4H), 1.23 (s, 6H) CFH367-C

δ 8.36 (t, J = 5.4 Hz, 1H), 7.88 (d, J = 3.0 Hz, 1H), 7.74 (d, J = 3.0Hz, 1H), 3.41 (q, J = 6.6 Hz, 2H), 3.31-3.36 (m, 1H), 2.16 (t, J = 6.6Hz, 2H), 1.64-1.69 (m, 4H), 1.29-1.34 (m, 2H), 0.82-0.86 (m, 2H) CFH409

δ 7.90 (d, J = 3.3 Hz, 1H), 7.74 (d, J = 3.3 Hz, 1H), 3.93-4.01 (m, 1H),3.40 (t, J = 6.6 Hz, 2H), 2.16 (t, J = 7.2 Hz, 2H), 1.77-1.89 (m, 2H),1.66-1.69 (m, 6H), 1.29-1.57 (m, 6H) CFH409-A

δ 8.29 (t, J = 5.4 Hz, 1H), 7.89 (s, 1H), 7.85 (d, J = 3.3 Hz, 1H), 7.70(d, J = 3.3 Hz, 1H), 3.31 (q, J = 6.3 Hz, 2H), 3.15-3.17 (m, 1H), 2.16(t, J = 6.0 Hz, 2H), 1.65-1.68 (m, 4H), 1.16- 1.22 (m, 2H), 1.06 (d, J =6.6 Hz, 3H), 1.02 (d, J = 6.6 Hz, 3H), 0.92-0.96 (m, 2H) CFH403

δ 8.48 (t, J = 5.4 Hz, 1H), 7.93 (d, J = 3.0 Hz, 1H), 7.89 (s, 1H), 7.77(d, J = 3.0 Hz, 1H), 7.59 (d, J = 6.6 Hz, 2H), 7.42-7.44 (m, 3H), 3.41(q, J = 6.0 Hz, 2H), 2.12 (t, J = 6.6 Hz, 2H), 1.62-1.64 (m, 4H) CFH421

δ 8.51 (t, J = 5.4 Hz, 1H), 7.93 (d, J = 3.0 Hz, 1H), 7.89 (s, 1H), 7.78(d, J = 3.0 Hz, 1H), 7.62 (t, J = 5.1 Hz, 2H), 7.17 (t, J = 8.4 Hz, 2H),3.33 (q, J = 8.4 Hz, 2H), 2.13 (t, J = 6.3 Hz, 2H), 1.63-1.65 (m, 4H)CFH455

δ 8.52 (t, J = 5.4 Hz, 1H), 7.95 (d, J = 3.3 Hz, 1H), 7.82 (d, J = 3.3Hz, 1H), 7.53 (t, J = 8.7 Hz, 1H), 7.37 (d, J = 8.7 Hz, 1H), 7.18 (t, J= 8.1 Hz, 1H), 3.34 (q, J = 6.6 Hz, 2H), 2.12 (t, J = 6.6 Hz, 2H),1.62-1.64 (m, 4H) CFH437

δ 8.47 (t, J = 5.4 Hz, 1H), 7.94 (d, J = 3.0 Hz, 1H), 7.79 (d, J = 3.0Hz, 1H), 7.50 (t, J = 7.8 Hz, 2H), 7.39 (t, J = 7.8 Hz, 2H), 3.31 (q, J= 6.6 Hz, 2H), 1.99 (t, J = 5.4 Hz, 2H), 1.17-1.19 (m, 4H) CFH447

δ 8.48 (t, J = 5.4 Hz, 1H), 8.25 (d, J = 8.4 Hz, 1H), 7.97 (d, J = 7.8Hz, 1H), 7.91 (d, J = 3.0 Hz, 1H), 7.58 (t, J = 8.1 Hz, 2H), 7.53 (d, J= 3.0 Hz, 1H), 3.40 (q, J = 6.6 Hz, 2H), 2.44 (t, J = 6.9 Hz, 2H),1.63-1.72 (m, 4H) CFH448-P

δ 8.22 (d, J = 7.2 Hz, 2H), 7.90 (d, J = 3.0 Hz, 1H), 7.79 (d, J = 6.9Hz, 2H), 7.58 (t, J = 6.0 Hz, 1H), 7.53 (d, J = 3.0 Hz, 1H), 3.40 (q, J= 6.6 Hz, 2H), 2.34 (t, J = 6.9 Hz, 2H), 1.59-1.74 (m, 4H) CFH417

δ 8.43 (t, J = 5.4 Hz, 1H), 7.86 (d, J = 3.0 Hz, 1H), 7.73 (d, J = 3.0Hz, 1H), 7.23- 7.34 (m, 5H), 4.70 (s, 2H), 3.44 (q, J = 6.3 Hz, 2H),2.17 (t, J = 6.6 Hz, 2H), 1.65-1.69 (m, 4H) CFH430

δ 8.39 (d, J = 16.5 Hz, 1H), 7.94 (d, J = 3.0 Hz, 1H), 7.90 (s, 1H),7.79 (d, J = 3.0 Hz, 1H), 7.59 (d, J = 6.9 Hz, 2H), 7.32-7.38 (m, 3H),7.18 (d, J = 16.5 Hz, 1H), 3.43 (q, J = 6.9 Hz, 2H), 2.16 (t, J = 7.5Hz, 2H), 1.63-1.70 (m, 4H) CFH461

(CDCl₃) δ 7.84 (d, J = 3.0 Hz, 1H), 7.70 (t, J = 5.4 Hz, 1H), 7.41 (d, J= 3.0 Hz, 1H), 7.27-7.41 (m, 5H), 4.53 (s, 2H), 3.76 (t, J = 4.5 Hz,2H), 3.67 (t, J = 4.8 Hz, 2H), 3.34 (q, J = 5.1 Hz, 2H), 2.18 (t, J =6.6 Hz, 2H), 1.61-1.69 (m, 4H) CFH324-C

δ 7.89 (d, J = 3.3 Hz, 1H), 7.74 (d, J = 3.3 Hz, 1H), 4.04 (s, 2H),3.30-3.32 (m, 1H), 1.29-1.34 (m, 2H), 0.83-0.85 (m, 2H) CFH338

δ 8.37 (t, J = 5.4 Hz, 1H), 7.89 (s, 1H), 7.85 (d, J = 3.0 Hz, 1H), 7.70(d, J = 3.0 Hz, 1H), 3.68 (q, J = 5.7 Hz, 2H), 3.25-3.31 (m, 1H), 2.46(t, J = 6.3 Hz, 2H), 1.28-1.31 (m, 2H), 0.79-0.81 (m, 2H) CFH352-C

δ 8.34 (t, J = 5.4 Hz, 1H), 7.88 (s, 1H), 7.85 (d, J = 3.0 Hz, 1H), 7.71(d, J = 3.0 Hz, 1H), 3.44 (q, J = 6.3 Hz, 2H), 3.23-3.28 (m, 1H), 2.21(t, J = 7.2 Hz, 2H), 1.91-1.99 (m, 2H), 1.27- 1.33 (m, 2H), 0.77-0.83(m, 2H) CFH381-C

δ 8.24 (t, J = 5.4 Hz, 1H), 7.89 (s, 1H), 7.85 (d, J = 3.0 Hz, 1H), 7.71(d, J = 3.0 Hz, 1H), 3.39 (q, J = 6.0 Hz, 2H), 3.24-3.29 (m, 1H), 2.13(t, J = 7.5 Hz, 2H), 1.63-1.71 (m, 4H), 1.42- 1.44 (m, 2H), 1.27-1.33(m, 2H), 0.78-0.83 (m, 2H) CFH395-C

δ 8.27 (t, J = 5.4 Hz, 1H), 7.89 (s, 1H), 7.87 (d, J = 3.0 Hz, 1H), 7.72(d, J = 3.0 Hz, 1H), 3.40 (q, J = 6.6 Hz, 2H), 3.25-3.29 (m, 1H), 2.11(t, J = 7.2 Hz, 2H), 1.63-1.67 (m, 4H), 1.40- 1.43 (m, 4H), 1.28-1.35(m, 2H), 0.80-0.85 (m, 2H) CFH409-C

δ 8.25 (t, J = 5.4 Hz, 1H), 7.89 (s, 1H), 7.87 (d, J = 3.0 Hz, 1H), 7.72(d, J = 3.0 Hz, 1H), 3.40 (q, J = 6.6 Hz, 2H), 3.25-3.28 (m, 1H), 2.09(t, J = 7.2 Hz, 2H), 1.62-1.65 (m, 4H), 1.33- 1.39 (m, 6H), 1.28-1.35(m, 2H), 0.79-0.86 (m, 2H) CFH397-2

δ 8.34 (t, J = 5.7 Hz, 1H), 7.90 (s, 1H), 7.55 (s, 1H), 3.38 (q, J = 6.6Hz, 2H), 3.20 (d, J = 6.9 Hz, 2H), 2.53 (s, 3H), 2.33 (t, J = 7.2 Hz,2H), 1.92-2.01 (m, 1H), 1.64-1.67 (m, 4H), 0.97 (d, J = 6.6 Hz, 6H)CFH381-B

δ7.89 (s, 1H), 7.26 (s, 1H), 3.41 (q, J = 6.0 Hz, 2H), 3.26-3.31 (m,1H), 2.44 (s, 3H), 2.15 (t, J = 6.0 Hz, 2H), 1.63-1.68 (m, 4H),1.28-1.31 (m, 2H), 0.80-0.82 (m, 2H) CFH381-M

δ7.89 (s, 1H), 7.54 (s, 1H), 3.41 (q, J = 6.0 Hz, 2H), 3.26-3.31 (m,1H), 2.54 (s, 3H), 2.16 (t, J = 6.0 Hz, 2H), 1.63-1.68 (m, 4H),1.28-1.31 (m, 2H), 0.79-0.81 (m, 2H) CFH395-M

δ7.90 (s, 1H), 3.40 (q, J = 6.0 Hz, 2H), 3.24-3.26 (m, 1H), 2.39 (s,3H), 2.30 (s, 3H), 1.99 (t, J = 5.4 Hz, 2H), 1.63-1.68 (m, 4H), 1.26-1.29 (m, 2H), 0.77-0.78 (m, 2H) CFH421-C

δ7.89 (s, 1H), 3.40 (q, J = 6.6 Hz, 2H), 3.25-3.30 (m, 1H), 2.84 (t, J =4.2 Hz, 2H), 2.76 (t, J = 4.2 Hz, 2H), 2.16 (t, J = 6.6 Hz, 2H),1.83-1.89 (m, 4H), 1.67-1.69 (m, 4H), 1.26-1.32 (m, 2H), 0.79-0.83 (m,2H) CFH407

δ 8.22 (s, 1H), 3.36 (q, J = 6.6 Hz, 2H), 3.21-3.24 (m, 1H), 2.90 (t, J= 6.6 Hz, 2H), 2.77 (t, J = 6.6 Hz, 2H), 2.49 (t, J = 6.3 Hz, 2H), 2.11(q, J = 6.6 Hz, 2H), 1.62-1.70 (m, 4H), 1.23- 1.26 (m, 2H), 0.74-0.76(m, 2H) CFH435

δ7.91 (s, 1H), 3.40 (q, J = 6.6 Hz, 2H), 3.25-3.28 (m, 1H), 2.94 (t, J =5.4 Hz, 2H), 2.90 (t, J = 5.1 Hz, 2H), 2.16 (t, J = 6.6 Hz, 2H),1.88-1.92 (m, 2H), 1.68-1.75 (m, 8H), 1.26-1.32 (m, 2H), 0.77-0.83 (m,2H) CFH449

δ7.90 (s, 1H), 3.40 (q, J = 6.6 Hz, 2H), 3.24-3.27 (m, 1H), 2.94 (t, J =5.4 Hz, 2H), 2.87 (t, J = 6.0 Hz, 2H), 2.15 (t, J = 6.3 Hz, 2H),1.69-1.77 (m, 8H), 1.37-1.44 (m, 4H), 1.21-1.24 (m, 2H), 0.78-0.81 (m,2H) CFH412-C

δ8.61 (s, 1H), 3.38 (q, J = 6.0 Hz, 2H), 3.28-3.31 (m, 1H), 2.15 (t, J =6.3 Hz, 2H), 1.63-1.68 (m, 4H), 1.22-1.29 (m, 2H), 0.71-0.73 (m, 2H)CFH407-C

δ7.83 (s, 1H), 7.12 (s, 1H), 3.33 (q, J = 6.3 Hz, 2H), 3.17-3.24 (m,1H), 2.10 (t, J = 5.7 Hz, 2H), 1.99-2.03 (m, 1H), 1.59-1.64 (m, 4H),1.20-1.23 (m, 2H), 0.88-0.92 (m, 2H), 0.81- 0.87 (m, 2H), 0.72-0.79 (m,2H) CFH449-H

δ7.89 (s, 1H), 3.35 (q, J = 6.6 Hz, 2H), 3.24-3.28 (m, 1H), 2.78 (t, J =4.2 Hz, 2H), 2.71 (t, J = 4.2 Hz, 2H), 2.15 (t, J = 6.6 Hz, 2H),1.89-1.98 (m, 4H), 1.61-1.69 (m, 4H), 1.32-1.38 (m, 4H), 1.25-1.28 (m,2H), 0.76-0.80 (m, 2H) CFH443-5

δ8.15 (s, 1H), 7.72 (d, J = 7.2 Hz, 2H), 7.45-7.47 (m, 3H), 3.44 (q, J =6.3 Hz, 2H), 3.31-3.35 (m, 1H), 2.16 (t, J = 7.2 Hz, 2H), 1.70- 1.75 (m,4H), 1.30-1.35 (m, 2H), 0.82-0.88 (m, 2H) CFH443-4

δ8.34 (s, 1H), 7.94 (d, J = 12 Hz, 2H), 7.35-7.43 (m, 3H), 3.41 (q, J =6.3 Hz, 2H), 3.27-3.31 (m, 1H), 2.17 (t, J = 7.2 Hz, 2H), 1.68- 1.73 (m,4H), 1.30-1.33 (m, 2H), 0.84-0.85 (m, 2H) CFH455-C

δ7.77 (s, 1H), 7.72 (d, J = 8.1 Hz, 2H), 7.41 (d, J = 7.8 Hz, 2H), 4.60(d, J = 6.0 Hz, 2H), 3.26-3.31 (m, 1H), 2.74 (s, 4H), 1.85 (s, 4H),1.21-1.26 (m, 2H), 0.74-0.78 (m, 2H) CFH401

δ7.87 (d, J = 3.3 Hz, 1H), 7.74 (d, J = 3.3 Hz, 1H), 7.72 (d, J = 8.4Hz, 2H), 7.47 (d, J = 7.8 Hz, 2H), 4.65 (s, 2H), 3.31-3.35 (m, 1H),1.30-1.33 (m, 2H), 0.83-0.85 (m, 2H) CFH403-1

δ7.91 (d, J = 8.7 Hz, 2H), 7.85 (d, J = 3.3 Hz, 1H), 7.83 (d, J = 8.4Hz, 2H), 7.77 (d, J = 3.3 Hz, 1H), 3.28 (d, J = 7.2 Hz, 2H), 1.96-2.05(m, 1H), 1.02 (d, J = 6.6 Hz, 6H) CFH429

δ 8.03 (s, 1H), 7.92 (d, J = 3.3 Hz, 1H), 7.78 (d, J = 3.3 Hz, 1H), 7.69(d, J = 7.5 Hz, 1H), 7.57 (d, J = 15.0 Hz, 1H), 7.34-7.39 (m, 2H), 6.49(d, J = 15.9 Hz, 1H), 3.28 (d, J = 7.2 Hz, 2H), 2.02- 2.04 (m, 1H), 1.01(d, J = 6.6 Hz, 6H) CFH394

(DMSO-d₆) δ 10.53 (s, 1H), 8.76 (s, 1H), 7.98 (d, J = 3.0 Hz, 1H), 7.93(d, J = 2.7 Hz, 1H), 4.49 (d, J = 13.2 Hz, 1H), 3.66 (d, J = 12.9 Hz,1H), 3.16 (t, J = 13.5 Hz, 1H), 2.95 (t, J = 12.3 Hz, 1H), 2.78 (d, J =6.9 Hz, 2H), 2.29-2.32 (m, 1H), 1.85- 1.90 (m, 1H), 1.70-1.83 (m, 1H),1.53-1.60 (m, 3H), 0.90 (d, J = 6.6 Hz, 6H) CFH412-4

δ 9.9 (s, 1H), 8.36 (s, 1H), 8.25 (s, 1H), 7.89 (s, 1H), 4.39 (t, J =7.2 Hz, 1H), 3.28 (q, J = 8.9 Hz, 2H), 2.11-2.21 (m, 1H), 1.83 (t, J =7.8 Hz, 2H), 1.04 (d, J = 6.9 Hz, 6H) CFH384

δ 9.09 (s, 1H), 8.36 (s, 1H), 8.26 (s, 1H), 7.89 (s, 1H), 4.46 (d, J =6.6 Hz, 1H), 3.85 (d, J = 6.6 Hz, 2H), 2.23-2.26 (m, 1H), 1.06 (d, J =6.9 Hz, 6H) CFH398-4

δ 9.08 (s, 1H), 8.36 (s, 1H), 8.28 (d, J = 9.0 Hz, 1H), 8.24 (s, 1H),7.89 (s, 1H), 4.39 (t, J = 7.2 Hz, 1H), 3.50 (q, J = 4.8 Hz, 2H), 2.34(t, J = 6.9 Hz, 2H), 2.15-2.22 (m, 1H), 1.03 (d, J = 6.6 Hz, 6H)CFH398-M

(DMSO-d₆) δ 10.55 (s, 1H), 8.86 (s, 1H), 8.54 (t, J = 5.4 Hz, 1H), 8.33(s, 1H), 8.23 (s, 1H), 8.01 (d, J = 9.0 Hz, 1H), 4.45 (t, J = 6.3 Hz,1H), 3.50 (d, J = 4.2 Hz, 2H), 2.73 (s, 3H), 2.10 (m, 1H), 0.94 (d, J =6.9 Hz, 3H), 0.89 (d, J = 6.9 Hz, 3H) CFH412-M

δ 8.27 (d, J = 8.4 Hz, 1H), 8.21 (s, 1H), 8.12 (s, 1H), 7.89 (s, 1H),4.38 (d, J = 6.0 Hz, 1H), 3.36 (t, J = 6.0 Hz, 2H), 2.76 (s, 3H), 2.34(t, J = 6.0 Hz, 2H), 2.15-2.21 (m, 1H), 1.02 (d, J = 6.6 Hz, 6H)CFH426-M

δ 8.34 (t, J = 6.0 Hz, 1H), 8.27 (d, J = 8.4 Hz, 1H), 8.21 (s, 1H), 8.12(s, 1H), 7.89 (s, 1H), 4.38 (d, J = 7.2 Hz, 1H), 3.25 (t, J = 6.0 Hz,2H), 2.76 (s, 3H), 2.17-2.20 (m, 1H), 2.14 (t, J = 7.2 Hz, 2H), 1.8-1.85(m, 2H), 1.03 (d, J = 6.6 Hz, 6H) CFH383-4

δ 9.06 (s, 1H), 8.51 (t, J = 5.4 Hz, 1H), 8.28 (s, 1H), 3.40 (q, J = 6.3Hz, 2H), 3.23 (d, J = 7.2 Hz, 2H), 2.16 (t, J = 6.6 Hz, 2H), 1.97- 2.02(m, 1H), 1.68-1.71 (m, 4H), 1.03 (d, J = 6.6 Hz, 6H) CFH397

δ 8.03 (s, 1H), 3.39 (q, J = 4.8 Hz, 2H), 3.21 (d, J = 7.2 Hz, 2H), 2.75(s, 3H), 2.16 (t, J = 6.6 Hz, 2H), 1.96-2.00 (m, 1H), 1.67- 1.69 (m,4H), 0.98 (d, J = 6.6 Hz, 6H) CFH326-4

(DMSO-d₆) δ 10.37 (s, 1H), 9.27 (s, 1H), 8.69 (s, 1H), 8.47 (t, J = 5.7Hz, 1H), 8.36 (s, 1H), 8.27 (s, 1H), 3.29 (q, J = 5.4 Hz, 2H), 1.98 (t,J = 6.9 Hz, 2H), 1.52 (m, 4H) CFH340-4

(DMSO-d₆) δ 10.36 (s, 1H), 8.68 (s, 1H), 8.43 (t, J = 6.0 Hz, 1H), 8.24(s, 1H), 8.14 (s, 1H), 3.30 (q, J = 6.9 Hz, 2H), 2.74 (s, 3H), 1.98 (t,J = 6.9 Hz, 2H), 1.52 (m, 4H) CFH456

δ 8.16 (s, 2H), 4.56 (d, J = 5.1 Hz, 2H), 3.43 (q, J = 5.7 Hz, 2H), 2.16(t, J = 7.2 Hz, 2H), 1.49-1.69 (m, 4H), 1.48 (s, 9H) CFH470

(DMSO-d₆) δ 10.28 (s, 1H), 8.70 (s, 1H), 8.45 (s, 1H), 8.25 (s, 1H),8.20 (s, 1H), 7.89 (t, J = 5.7 Hz, 1H), 4.45 (d, J = 5.1 Hz, 2H), 3.27(q, J = 5.7 Hz, 2H), 2.21 (t, J = 7.2 Hz, 2H), 1.49 (m, 6H), 1.42 (s,9H) CFH484

δ 8.16 (s, 1H), 7.76 (d, J = 3.3 Hz, 1H), 7.21 (d, J = 3.3 Hz, 1H), 4.56(s, 2H), 3.43 (q, J = 5.7 Hz, 2H), 2.16 (t, J = 7.2 Hz, 2H), 1.23- 1.59(m, 8H), 1.22 (s, 9H)

Example 2 of Preparation (Compound Number CFH494)

Compound 20 (90 mg, 0.24 mmol) was dissolved in THF/H₂O (5 mL, 4:1).NaOH (12 mg, 0.29 mmol) was added and stirred for 2 h at roomtemperature. The reaction mixture was acidified to pH=2-3 with 1Nhydrochloric acid, extracted by EtOAc (10 mL×3 times), washed with brine(20 mL), dried by anhydrous Na₂SO₄, and concentrated to obtain compound21 (85 mg, 98%, white solid). ¹H NMR (300 MHz, CD3OD) δ 7.86 (d, J=3.3Hz, 1H), 7.70 (d, J=3.3 Hz, 1H), 3.38 (t, J=6.3 Hz, 2H), 3.20 (d, J=7.2Hz, 2H), 2.36 (t, J=6.9 Hz, 2H), 1.89-2.00 (m, 1H), 1.66-1.68 (m, 4H),0.96 (d, J=6.6 Hz, 6H).

Compound 21 (93 mg, 0.25 mmol) was dissolved in dry CH₂Cl₂ (5 mL).Compound 22 (74 mg, 0.35 mmol), HOBt (41 mg, 0.30 mmol), Et₃N (36 mg,0.35 mmol) were added in respectively and stirred for 10 min at 0° C.,then EDCI (82 mg, 0.43 mmol) was added in. The reaction was kept at roomtemperature overnight. The obtained reaction mixture was sequentiallywashed by 1N hydrochloric acid (10 mL), saturated sodium bicarbonatesolution (10 mL) and brine, dried by anhydrous Na₂SO₄, concentrated andpurified by flash chromotography (PE/EtOAc=2: 1-1:1) to obtain compound23 (74 mg, 52.5%, colorless transparent oil). ¹H NMR (300 MHz, CDCl₃) δ7.86 (d, J=3.3 Hz, 1H), 7.66 (t, J=5.4 Hz, 1H), 7.52 (d, J=7.8 Hz, 1H),7.45 (d, J=3.3 Hz, 1H), 7.35 (d, J=7.8 Hz, 1H), 7.06-7.14 (m, 2H), 3.40(q, J=6.3 Hz, 2H), 3.26 (d, J=6.9 Hz, 2H), 2.38 (t, J=6.6 Hz, 2H),1.98-2.01 (m, 1H), 1.66-1.76 (m, 4H), 1.48 (s, 9H), 0.98 (d, J=6.6 Hz,6H).

Compound 23 (73 mg, 0.13 mmol) was dissolved in CH₂Cl₂ (2 mL), and thenEtOAc solution of 1N hydrochloric acid (2 mL) was added and stirred for10 min at room temperature. Product CFH494 (60 mg, 93.8%, white solid)was obtained after concentration. ¹H NMR (300 MHz, CD₃OD) δ 7.93 (d,J=3.3 Hz, 1H), 7.80 (d, J=3.3 Hz 1H), 7.47-7.52 (m, 2H), 7.35-7.42 (m,3H), 3.45 (q, J=6.6 Hz, 2H), 3.31 (d, J=7.2 Hz, 2H), 2.59 (t, J=6.6 Hz,2H), 1.81-2.00 (m, 1H), 1.74-1.79 (m, 4H), 0.98 (d, J=6.6 Hz, 6H).

The following compounds were synthesized using the same method as above:

Compound Structural formula ¹H NMR (CD₃OD, 300 MHz) data CHF494

δ 7.93 (d, J = 3.3 Hz, 1H), 7.80 (d, J = 3.3 Hz, 1H), 7.47-7.52 (m, 2H),7.35- 7.42 (m, 3H), 3.45 (q, J = 6.6 Hz, 2H), 3.31 (d, J = 7.2 Hz, 2H),2.59 (t, J = 6.6 Hz, 2H), 1.81-2.00 (m, 1H), 1.74-1.79 (m, 4H), 0.98 (d,J = 6.6 Hz, 6H) CFH508

δ 7.92 (d, J = 3.3 Hz, 1H), 7.79 (d, J = 3.3 Hz, 1H), 7.40-7.49 (m, 4H),7.34 (t, J = 7.8 Hz, 1H), 3.36 (t, J = 6.9 Hz, 2H), 3.19 (d, J = 7.2 Hz,2H), 2.52 (t, J = 7.2 Hz, 2H), 1.92-1.96 (m, 1H), 1.76-1.81 (m, 2H),1.65-1.73 (m, 2H), 1.44- 1.48 (m, 2H), 0.93 (d, J = 6.6 Hz, 6H) CFH522

δ 7.88 (d, J = 3.3 Hz, 1H), 7.73 (d, J = 3.3 Hz, 1H), 7.47-7.49 (m, 2H),7.31- 7.43 (m, 2H), 4.06 (t, J = 6.6 Hz, 2H), 3.05 (d, J = 6.9 Hz, 2H),2.51 (t, J = 7.2 Hz, 2H), 1.98-2.05 (m, 1H), 1.78-1.86 (m, 4H),1.53-1.67 (m, 4H), 1.03 (d, J = 6.6 Hz, 6H) CFH500

(CDCl₃) δ 8.71 (s, 1H), 8.66 (s, 1H), 7.87 (d, J = 3.3 Hz, 1H), 7.63 (t,J = 5.4 Hz, 1H), 7.45 (d, J = 3.3 Hz, 1H), 7.41 (d, J = 6.6 Hz, 1H),7.32 (d, J = 5.7 Hz, 1H), 7.13-7.15 (m, 2H), 3.45 (q, J = 6.6 Hz, 2H),3.27 (d, J = 7.2 Hz, 2H), 2.38 (t, J = 6.6 Hz, 2H), 2.10 (s, 3H),1.97-2.01 (m, 1H), 1.68- 1.76 (m, 4H), 0.99 (d, J = 6.6 Hz, 6H) CFH514

(CDCl₃) δ 8.79 (s, 2H), 7.87 (d, J = 3.0 Hz, 1H), 7.58 (t, J = 5.4 Hz,1H), 7.44 (d, J = 3.3 Hz, 1H), 7.31-7.36 (m, 2H), 7.11-7.14 (m, 2H),3.40 (q, J = 6.9 Hz, 2H), 3.28 (d, J = 6.9 Hz, 2H), 2.32 (t, J = 7.5 Hz,2H), 2.10 (s, 3H), 2.00-2.07 (m, 1H), 1.60-1.73 (m, 4H), 1.38-1.46 (m,2H), 1.00 (d, J = 6.6 Hz, 6H) CFH325

δ 7.74 (d, J = 3.0 Hz, 1H), 7.58 (d, J = 3.0 Hz, 1H), 4.00 (d, J = 5.1Hz, 2H), 3.07 d, J = 7.2 Hz, 2H), 1.82-1.88 (m, 1H), 0.84 (d, J = 6.6Hz, 6H) CFH339-A

δ 7.86 (d, J = 3.0 Hz, 1H), 7.70 (d, J = 3.0 Hz, 1H), 3.62 (t, J = 6.6Hz, 2H), 3.20 (d, J = 7.2 Hz, 2H), 2.63 (t, J = 6.6 Hz, 2H), 1.89-2.01(m, 1H), 0.96 (d, J = 6.6 Hz, 6H) CFH353

δ 7.89 (d, J = 3.3 Hz, 1H), 7.72 (d, J = 3.3 Hz, 1H), 3.43 (t, J = 6.9Hz, 2H), 3.23 (d, J = 6.9 Hz, 2H), 2.41 (t, J = 7.5 Hz, 2H), 1.98-2.03(m, 1H), 1.89-1.96 (m, 2H), 0.99 (d, J = 6.6 Hz, 6H) CFH367-A

δ 7.86 (d, J = 3.3 Hz, 1H), 7.70 (d, J = 3.3 Hz, 1H), 3.38 (t, J = 6.3Hz, 2H), 3.20 (d, J = 7.2 Hz, 2H), 2.36 (t, J = 6.9 Hz, 2H), 1.89-2.00(m, 1H), 1.66-1.68 (m, 4H), 0.96 (d, J = 6.6 Hz, 6H) CFH381-A

δ 7.84 (d, J = 3.3 Hz, 1H), 7.67 (d, J = 3.3 Hz, 1H), 3.33 (t, J = 6.9Hz, 2H), 3.17 (d, J =6.9 Hz, 2H), 2.15 (t, J = 7.5 Hz, 2H), 1.86-1.97(m, 1H) , 1.50-1.67 (m, 4H), 1.33-1.43 (m, 2H), 0.93 (d, J = 6.6 Hz, 6H)CFH381-A

δ7.83 (d, J = 3.0 Hz, 1H), 7.43 (d, J = 3.0 Hz, 1H), 3.82 (q, J = 7.2Hz, 2H), 3.13 (d, J = 6.9 Hz, 2H), 2.29 (t, J = 7.5 Hz, 2H), 1.91-1.98(m, 1H), 1.82-1.89 (m, 2H), 1.54-1.60 (m, 2H), 1.20- 1.35 (m, 4H), 0.93(d, J = 6.6 Hz, 6H) CFH410-A

δ 8.33 (t, J = 5.7 Hz, 1H), 7.88 (d, J = 3.0 Hz, 1H), 7.72 (d, J = 3.0Hz, 1H), 3.36 (q, J = 6.9 Hz, 2H), 3.26 (d, J = 7.5 Hz, 2H), 2.26 (t, J= 7.2 Hz, 2H), 1.93-2.00 (m, 1H), 1.58-1.61 (m, 4H), 1.34-1.37 (m, 6H),0.99 (d, J = 6.6 Hz, 6H) CFH379

δ 7.89 (d, J = 3.3 Hz, 1H), 7.71 (d, J = 3.3 Hz, 1H), 4.49 (d, J = 13.2Hz, 1H), 3.77 (d, J = 13.8 Hz, 1H), 3.07-3.26 (m, 2H), 2.84 (d, J = 7.2Hz, 2H), 2.63-2.70 (m, 1H), 2.04-2.08 (m, 1H), 1.87-1.99 (m, 2H),1.66-1.80 (m, 2H), 0.97 (d, J = 6.6 Hz, 6H)

Example 3 of Preparation (Compound Number CFH412)

Compound 24 (500 mg, 1.87 mmol) was dissolved in dry CH₂Cl₂ (15 mL) andthen compound 25 (754 mg, 1.87 mmol), EDCI (488 mg, 2.80 mmol), DMAP (23mg, 0.19 mmol) were sequentially added in. The reaction was kept at roomtemperature overnight. The reaction mixture was sequentially washed withsaturated sodium bicarbonate solution (10 mL) and brine. The aqueousphase was extracted with CHCl₃ (10 mL×2 times). The combination oforganic phases was dried with anhydrous Na₂SO₄, concentrated andpurified by column chromatography (PE/EtOAc=2: 1-1:1) to obtain product26 (640 mg, 52.5%, yellow grease). ¹H NMR (300 MHz, CDCl₃) δ 7.78 (d,J=3.0 Hz, 1H), 7.51 (t, J=6.3 Hz, 1H), 7.35 (d, J=8.1 Hz, 6H), 7.29 (d,J=3.0

Hz, 1H), 7.11-7.23 (m, 9H), 6.15 (t, J=6.0 Hz, 1H), 3.32 (q, J=6.0 Hz,2H), 3.22 (d, J=7.2 Hz, 2H), 3.05 (s, 2H), 2.96 (q, J=6.0 Hz, 2H),1.94-1.96 (m, 1H), 1.44-1.46 (m, 2H), 1.35-1.40 (m, 2H), 0.94 (d, J=6.6Hz, 6H).

Compound 26 (320 mg, 0.5 mmol) was dissolved in CH₂Cl₂ (5 mL). Et₃SiH(188 mg, 1.65 mmol) and trifluoroacetic acid (5.6 g, 0.05 mol) wereadded in and stirred for 2 h at room temperature. The reaction mixturewas concentrated under reduced pressure and purified by silica gelcolumn chromotography (PE/EA=1:1-1:5) to obtain product CFH412 (150 mg,74.4%, pale yellow grease). ¹H NMR (300 MHz, CDCl₃) δ 7.78 (d, J=3.0 Hz,1H), 7.59 (t, J=5.7 Hz, 1H), 7.38 (d, J=3.0 Hz 1H), 7.31 (t, J=5.7 Hz,1H), 3.36 (q, J=6.3 Hz, 2H), 3.26 (q, J=5.7 Hz, 2H), 3.19 (s, 2H), 3.17(d, J=7.2 Hz, 2H), 1.86-1.97 (m, 1H), 1.47-1.58 (m, 4H), 0.89 (d, J=6.6Hz, 6H).

The following compounds were synthesized using the same method as above:

Compounds Structural formula ¹H NMR (CDCl₃, 300 MHz) data CFH398-S

δ 7.81 (d, J = 3.0 Hz, 1H), 7.76 (t, J = 6.6 Hz, 1H), 7.57 (t, J = 5.7Hz, 1H), 7.41 (d, J = 3.0 Hz, 1H), 3.44 (q, J = 6.3 Hz, 2H), 3.31 (q, J= 5.7 Hz, 2H), 3.23 (s, 2H), 3.20 (d, J = 6.9 Hz, 2H), 1.91-1.96 (m,1H), 1.73-1.77 (m, 2H), 0.92 (d, J = 6.6 Hz, 6H) CFH412

δ 7.78 (d, J = 3.0 Hz, 1H), 7.59 (t, J = 5.7 Hz, 1H), 7.38 (d, J = 3.0Hz, 1H), 7.31 (t, J = 5.7 Hz, 1H), 3.36 (q, J = 6.3 Hz, 2H), 3.26 (q, J= 5.7 Hz, 2H), 3.19 (s, 2H), 3.17 (d, J = 7.2 Hz, 2H), 1.86-1.97 (m,1H), 1.47-1.58 (m, 4H), 0.89 (d, J = 6.6 Hz, 6H) CFH426

δ 7.74 (d, J = 3.0 Hz, 1H), 7.52 (t, J = 5.4 Hz, 1H), 7.36 (d, J = 3.0Hz, 1H), 7.10 (t, J = 5.7 Hz, 1H), 3.29 (q, J = 6.6 Hz, 2H), 3.17 (q, J= 5.7 Hz, 2H), 3.15 (s, 2H), 3.14 (d, J = 7.5 Hz, 2H), 1.85-1.93 (m,1H), 1.44-1.55 (m, 4H), 1.29-1.31 (m, 2H), 0.86 (d, J = 6.6 Hz, 6H)CFH440

δ 7.72 (d, J = 3.3 Hz, 1H), 7.49 (t, J = 5.7 Hz, 1H), 7.34 (d, J = 3.3Hz, 1H), 7.14 (t, J = 5.7 Hz, 1H), 3.25 (q, J = 6.6 Hz, 2H), 3.13 (q, J= 5.7 Hz, 2H), 3.10 (s, 2H), 3.09 (d, J = 7.2 Hz, 2H), 1.81-1.94 (m,1H), 1.37-1.47 (m, 4H), 1.06-1.11 (m, 4H), 0.83 (d, J = 6.6 Hz, 6H)CFH367-S

δ 7.99 (dd, J = 17.4, 11.1 Hz, 1H), 7.92 (d, J = 3.0 Hz, 1H), 7.73 (t, J= 3.6 Hz, 1H), 7.51 (d, J = 3.0 Hz, 1H), 5.78 (d, J = 17.4 Hz, 1H), 5.53(d, J = 11.1 Hz, 1H), 3.54 (q, J = 6.6 Hz, 2H), 3.40 (q, J = 6.0 Hz,2H), 3.29 (d, J = 9.0 Hz, 2H), 1.77-1.87 (m, 2H) CFH397-S

δ 7.90 (d, J = 3.0 Hz, 1H), 7.79 (t, J = 6.6 Hz, 1H), 7.61 (s, 1H), 7.47(d, J = 3.0 Hz, 1H), 3.53 (q, J = 6.3 Hz, 2H), 3.39 (q, J = 5.7 Hz, 2H),3.28 (d, J = 8.7 Hz, 2H), 2.04 (s, 6H), 1.77-1.85 (m, 2H) CFH383-S

δ 7.88 (d, J = 3.3 Hz, 1H), 7.69-7.73 (m, 1H), 7.46 (d, J = 3.3 Hz, 1H),5.28-6.33 (m, 1H), 3.50 (q, J = 6.6 Hz, 2H), 3.37 (q, J = 6.3 Hz, 2H),3.25 (d, J = 9.0 Hz, 2H), 1.82-1.94 (m, 3H), 1.75-1.80 (m, 2H) CFH411

δ 7.69 (d, J = 3.3 Hz, 1H), 7.41 (d, J = 10.5 Hz, 1H), 7.27 (d, J = 3.3Hz, 1H), 6.07 (dd, J = 15.9, 6.9 Hz, 1H), 3.32 (q, J = 5.4 Hz, 2H), 3.19(q, J = 5.4 Hz, 2H), 3.08 (d, J = 8.7 Hz, 2H), 2.31-2.38 (m, 1H),1.60-1.64 (m, 2H), 0.89 (d, J = 7.2 Hz, 6H)

Embodiment of Preparation 4 (Compound Number CFH538)

Compound 27 (100 mg, 0.47 mmol) was dissolved in dry DMF (5 mL) and themixture was cooled to 0° C. in an ice-bath. EDCI (108 mg, 0.57 mmol) andHOBt (76 mg, 0.57 mmol) were added in and stirred for 10 min at 0° C.Then, compound 28 (81 mg, 0.52 mmol) and i-Pr₂NEt (91 mg, 0.7 μmol) wereadded in and stirred for 12 h at room temperature. The obtained reactionmixture was diluted with 20 mL of water, extracted by EtOAc (15 mL×3times). The combined organic phases was washed by brine (20 mL) and thendried by anhydrous Na₂SO₄. After removal of the solvent, product 29 (129mg, 77.9%, colorless grease) was obtained after purification by silicagel column chromatography (PE/EtOAc=3:1). ¹H NMR (300 MHz, CDCl₃) δ 8.83(s, 1H), 8.10 (s, 1H), 8.05 (s, 1H), 7.81 (d, J=9.0 Hz, 1H), 5.87 (dddd,J=17.1, 10.5, 5.7, 5.1 Hz, 1H), 5.31 (d, J=17.1 Hz, 1H), 5.23 (d, J=10.5Hz, 1H), 4.73 (dd, J=9.3, 5.1 Hz, 1H), 4.65 (dd, J=17.1, 5.7 Hz, 2H),2.23-2.33 (m, 1H), 0.96 (t, J=6.9 Hz, 6H).

Compound 29 (55 mg, 0.16 mmol) was dissolved in redistilled toluene (5mL). To this solution was added a toluene solution (1 mL) of Grubbssecond generation catalyst (66 mg, 0.08 mmol), and toluene (1 mL)solution of compound 30 (100 mg, 0.48 mmol). The reaction mixture washeated to 110° C. and refluxed for 12 h. The obtained reaction mixturewas concentrated and then purified by silica gel column chromatography(PE/EtOAc=2:1) to obtain product CFH538 (23 mg, 50%, pale yellowgrease). Crude material 29 (25 mg) was recycled. ¹H NMR (300 MHz, CDCl₃)δ 8.86 (s, 1H), 8.13 (s, 1H), 8.11 (s, 1H), 7.83 (d, J=9.0 Hz, 1H), 5.74(dt, J=15.3, 6.6 Hz, 1H), 5.63 (dt, J=13.2, 6.0 Hz, 1H), 4.74 (dd,J=9.0, 5.4 Hz, 1H), 4.66 (dd, J=15.3, 5.4 Hz, 2H), 2.91 (t, J=7.5 Hz,2H), 2.52 (t, J=7.5 Hz, 2H), 2.35-2.43 (m, 2H), 2.22-2.32 (m, 1H),1.60-1.67 (m, 2H), 1.19-1.26 (m, 8H), 1.04 (t, J=6.3 Hz, 6H), 0.86 (t,J=6.9 Hz, 3H).

The following compounds were synthesized using the same method as above:

Compounds Structural formula ¹H NMR (CDCl₃, 300 MHz) data CFH550

δ 7.87 (d, J = 3.0 Hz, 1H), 7.48 (t, J = 4.5 Hz, 1H), 7.44 (d, J = 3.0Hz, 1H), 5.47 (ddd, J = 12.6, 6.3, 6.3 Hz, 1H), 5.37 (ddd, J = 10.5,6.0, 6.0 Hz, 1H), 3.43 (q, J = 7.2 Hz, 2H), 3.28 (d, J = 7.5 Hz, 2H),2.88 (t, J = 7.5 Hz, 2H), 2.51 (t, J = 7.5 Hz, 2H), 2.21 (m, 1H), 1.99(m, 4H), 1.61 (m, 4H), 1.38 (m, 4H), 1.23 (m, 8H), 0.89 (d, J = 6.9 Hz,6H), 0.83 (t, J = 6.6 Hz, 3H) CFH384-S

δ 9.38 (s, 1H), 7.89 (d, J = 3.0 Hz, 1H), 7.46 (d, J = 3.0 Hz, 1H), 3.80(q, J = 6.3 Hz, 2H), 3.66 (t, J = 6.3 Hz, 2H), 3.51 (d, J = 7.2 Hz, 2H),2.15 (m, 1H), 1.78 (m, 2H), 1.61 (m, 2H), 1.48 (m, 4H), 0.86 (d, J = 6.6Hz, 6H) CFH412-S

δ 8.87 (s, 1H), 8.14 (s, 1H), 8.09 (s, 1H), 7.84 (d, J = 9 Hz, 1H), 5.78(dt, J = 15.3, 6.6 Hz, 1H), 5.68 (dt, J = 12.3, 6.3 Hz, 1H), 4.76 (dd, J= 8.7, 4.8 Hz, 1H), 4.65 (dd, J = 15.3, 5.4 Hz, 2H), 2.58 (q, J = 7.5Hz, 2H), 2.39-2.44 (m, 2H), 2.31-2.37 (m, 1H), 1.04 (d, J = 6.3 Hz, 6H)CFH426-S

δ 8.11 (s, 1H), 7.89 (s, 1H), 7.86 (d, J = 6.3 Hz, 1H), 5.77 (ddd, J =15.3, 6.3, 6.3 Hz, 1H), 5.65 (ddd, J = 14.4, 6.6, 6.6 Hz, 1H), 4.74 (dd,J = 9.0, 4.8 Hz, 1H), 4.64 (dd, J = 15.3, 6.9 Hz, 2H), 2.77 (s, 3H),2.57 (q, J = 7.5 Hz, 2H), 2.36-2.43 (m, 2H), 2.28-2.34 (m, 1H), 1.05 (d,J = 6.3 Hz, 3H) CFH538

δ 8.86 (s, 1H), 8.13 (s, 1H), 8.11 (s, 1H), 7.83 (d, J = 9.0 Hz, 1H),5.74 (dt, J = 15.3, 6.6 Hz, 1H), 5.63 (dt, J = 13.2, 6.0 Hz, 1H),4.74(dd, J = 9.0, 5.4 Hz, 1H), 4.66 (dd, J = 15.3, 5.4 Hz, 2H), 2.91 (t,J = 7.5 Hz, 2H), 2.52 (t, J = 7.5 Hz, 2H), 2.35-2.43 (m, 2H), 2.22-2.32(m, 1H), 1.60-1.67 (m, 2H), 1.19-1.26 (m, 8H), 1.04 (t, J = 6.3 Hz, 6H),0.86 (t, J = 6.9 Hz, 3H) CFH552

δ 8.10 (s, 1H), 7.89 (s, 1H), 7.82 (d, J = 9.0 Hz, 1H), 5.75 (ddd, J =15.3, 6.6, 6.6 Hz, 1H), 5.67 (ddd, J = 14.4, 7.2, 7.2 Hz, 1H), 4.74(dd,J = 9.0, 5.1 Hz, 1H), 4.61 (dd, J = 15.3, 4.5 Hz, 2H), 2.90 (t, J = 7.2Hz, 2H), 2.77 (s, 3H), 2.51 (t, J = 7.2 Hz, 2H), 2.31-2.36(m, 2H),2.27-2.30 (m, 1H), 1.61-1.66 (m, 2H), 1.19-1.27 (m, 8H), 1.02 (t, J =6.6 Hz, 6H), 0.86 (t, J = 7.2 Hz, 3H)

Example 5 of Preparation (Compound Number CFH325-B)

Compound 31 (1 g, 7.86 mmol) was dissolved in dry DME (30 mL).Lawesson's reagent (1.6 g, 3.9 mmol) was added in with the protection ofN₂. After 12 h of reaction at room temperature, Compound 32 (1.0 g,88.8%, white solid), was obtained after filtration, removal of solvent,and purification by silica gel column chromatography(PE/EtOAc=10:1-2:1). Compound 32 was used for next reaction directly,

Compound 32 (500 mg, 3.49 mmol) was dissolved in dry DME (30 mL). KHCO₃(2.1 g, 21 mmol) was added and stirred for 10 min at room temperature.Compound 33 (1.5 g, 7.68 mmol) was added dropwise. After 1 h ofreaction, the reaction mixture was cooled in an ice-bath and then addeddropwise trifluoroacetic anhydride (2.2 g, 10.48 mmol) and DME (10 mL)solution of 2,6-lutidine (1.87 g, 17.46 mmol). The reaction was kept at0° C. for 1 h, and kept overnight after rising to room temperature.After the reaction of compound 32 was completed which was detected byTLC, the solvent was removed by evaporating under reduced pressure andthen diluted with EtOAc (50 mL). The organic phase was sequentiallywashed with 1N hydrochloric acid (20 mL), saturated sodium bicarbonate(20 mL) and brine (20 mL), and dried with anhydrous Na₂SO₄. Compound 34(647 mg of 77.4%, pale yellow solid) was obtained after purification bysilica gel chromatography (PE/EtOAc=4: 1-2:1). ¹H NMR (300 MHz, CDCl₃) δ8.00 (s, 1H), 7.50 (d, J=3.9 Hz, 1H), 7.36 (d, J=5.1 Hz, 1H), 7.00 (dd,J=4.8, 3.9 Hz, 1H), 4.34 (q, J=7.2 Hz, 2H), 1.33 (t, J=7.2 Hz, 3H)

Compound 34 (447 mg, 1.87 mmol) was dissolved in EtOH/H₂O (20/5 mL).NaOH (149 mg, 3.74 mol) solid was added in at 0° C. and was stirred atroom temperature overnight. After the completion of the reaction, EtOHwas removed under reduced pressure, diluted with H₂O and acidified topH=2 by 1N hydrochloric acid and extraction by EtOAc (20 mL×3 times).The collected organic phase was washed by brine (20 mL) and then driedwith anhydrous Na₂SO₄. Compound 35 was obtained by concentration, whichwas used directly for the next reaction.

Compound 35 (110 mg, 0.52 mmol) was dissolved in dry DMF (5 mL), thencooled to 0° C. in an ice-bath. EDCI (150 mg, 0.78 mmol) and HOBt (106mg, 0.78 mmol) was added in and stirred for 10 min at 0° C. Compound 18(96 mg, 0.57 mmol) and i-Pr₂NEt (134 mg, 1.04 mmol) were added in andstirred for 12 h at room temperature. The obtained mixture was dilutedwith 20 mL water, then extracted by EtOAc (15 mL×3 times). The combinedorganic phases was washed by 1N hydrochloric acid (15 ml), saturatedsodium bicarbonate solution (15 ml) and brine (20 mL) in sequence, andthen dried with anhydrous Na₂SO₄. After removal of solvent byevaporating under reduced pressure, the obtained residue was purified bysilica gel column chromatography (PE/EtOAc=2:1) to obtain productcompound 36 (146 mg, 86.4%, pale yellow grease). ¹H NMR (300 MHz, CDCl₃)δ 7.95 (s, 1H), 7.47 (d, J=3.9 Hz, 1H), 7.39 (d, J=5.1 Hz, 1H), 7.03(dd, J=5.1, 3.9 Hz, 1H), 3.62 (s, 3H), 3.42 (q, J=6.3 Hz, 2H), 2.31 (t,J=6.6 Hz, 2H), 1.65-1.71 (m, 4H).

Hydroxylamine hydrochloride (215 mg, 3.1 mmol) was suspended in MeOH (15mL), and then KOH (260 mg, 4.65 mmol) was added and stirred for 5 min.The insoluble substance was removed by filtration and the filtrate wascollected for next use. After compound 36 (100 mg, 0.31 mmol) wasdissolved in anhydrous MeOH (10 ml), freshly-prepared above-mentionedMeOH solution of hydroxylamine hydrochloride was added in and stirredfor 1 h at room temperature. Reaction endpoint was determined by TLC.The obtained solution was diluted with EtOAc, then neutralized to pH=5-6by 1N hydrochloric acid. After removal of solvent by evaporating underreduced pressure, the obtained mixture was sequentially extracted byEtOAc (15 mL×3 times), and washed by brine (10 mL), dried by anhydrousNa₂SO₄ and concentrated to obtain the crude product which was purifiedby column chromatography (CHCl₃/MeOH=20:1-10:1) to obtain productCFH325-B (86 mg, 86%, white solid). ¹H NMR (300 MHz, CD₃OD) δ 8.09 (s,1H), 7.87 (s, 1H), 7.66 (d, J=3.9 Hz, 1H), 7.60 (d, J=5.1 Hz, 1H), 7.14(dd, J=3.9, 5.1 Hz, 1H), 3.42 (q, J=6.3 Hz, 2H), 2.16 (t, J=6.6 Hz, 2H),1.68-1.71 (m, 4H).

Testing Example of Bio-Experiment Testing Example 1 Experiment ofDetecting the Inhibitory Activity for Histone Deacetylase 1, 3, 6(HDAC1, 3, 6)

1. Experimental Objective:

The objective is to detect the inhibitory activity of the compoundsagainst recombinant human histone deacetylase 1, 3, 6.

2. Source of the Material:

Human HDAC1, HDAC3 and HDAC6 are obtained from of the baculovirusexpression system.

3. Principle:

Enzyme activity of HDAC1, 3 is determined using the substrateAc-Lys-Tyr-Lys(Ac)-AMC while the enzyme activity of HDAC6 is assayedusing the substrate Boc-lys(Ac)-AMC. The reaction is carried out inflat-bottom. 96-well or 384-well microplates. After the substrate wasdeacetylated by HDACs, the product AMC which was obtained fromhydrolysis by trypsin can generate fluorescence signal. Measurement istaken in using an multilabel plate reader a 355 nm excitation filter anda 460 nm emission filter. The initial rate of fluorescence shouldaccurately reflect the rate of product formation and enzyme activity.

4. Experimental Process

Sample processing: The sample was dissolved in DMSO and kept at lowtemperature. DMSO in the final system is limited under a lowconcentration which won't affect the enzyme activity.

Data processing and results: The single concentration of the compounds,for example, 20 μg/ml, was used in the preliminary screening and theactivity of the sample was tested. As for the samples that exhibit acertain activity, e.g. the inhibition ratio is greater than 50%, theactivity-dose dependency relationship, IC₅₀/EC₅₀ value, were determined,which could be obtained by the nonlinear fitting of the activity againstthe concentration of the samples. The calculation software for thenonlinear fitting is Graphpad Prism 4, and the model for the fitting issigmoidal dose-response (variable slope). For the mostinhibitor-screening models, the bottom and the top of the fitted curveis set as 0 and 100. Generally, all measurements were duplicated (n≧2),and the results are indicated as mean±SD (Standard Deviation) or SE(standard error). A well-reported HDAC pan-inhibitor, SAHA (Vorinostat)was used as a positive control in each measurement.

5. Experimental Results:

Sample HDAC1 HDAC3 HDAC6 Sample HDAC1 HDAC3 HDAC6 No. IC₅₀(μM) IC₅₀(μM)IC₅₀(μM) No. IC₅₀(μM) IC₅₀(μM) IC₅₀(μM) CFH326 0.212 0.107 1.110 CFH4010.042 0.030 0.020 CFH340-M 0.606 0.691 1.106 CFH403-1 1.020 0.687 0.295CFH355 0.031 0.256 0.938 CFH429 0.657 0.410 0.305 CFH369 0.341 0.3581.206 CFH394 NA NA NA CFH383 0.031 0.188 0.621 CFH412-4 2.587 1.63829.451  CFH382 0.047 0.102 1.217 CFH384 NA NA NA CFH340   NA^(a) NA NACFH398-4 9.593 3.136 NA CFH354 NA NA NA CFH398-M NA NA NA CFH368 NA NA19.511  CFH412-M 2.655 2.218 NA CFH396 0.035 0.086 0.497 CFH426-M 2.4531.577 NA CFH410 0.178 0.249 1.246 CFH383-4 1.102 0.352 3.159 CFH4240.021 0.073 0.575 CFH397 0.438 0.224 2.045 CFH381 0.173 0.194 0.176CFH326-4 0.816 0.193 1.331 CFH395 0.013 0.142 0.167 CFH340-4 0.535 0.6151.194 CFH367 0.022 0.106 0.376 CFH456 0.897 0.895 1.156 CFH352 0.1160.384 2.295 CFH470 0.121 0.174 0.566 CFH399 1.574 2.203 9.536 CFH4841.684 1.178 1.424 CFH367-C 0.065 0.262 0.785 CFH494 5.911 3.814 NACFH409 0.347 0.325 0.748 CFH508 0.984 4.307 NA CFH409-A 0.628 0.2760.188 CFH522 15.343  NA NA CFH403 0.114 0.377 0.467 CFH500 NA NA NACFH421 0.204 0.314 0.501 CFH514 NA NA NA CFH455 0.488 0.587 4.991 CFH325NA NA NA CFH437 0.165 0.453 5.178 CFH339-A NA NA NA CFH447 0.270 0.3640.473 CFH353 NA NA NA CFH448-P 0.105 0.201 1.067 CFH367-A  NT^(b) NT NTCFH417 0.058 0.079 1.746 CFH381-A NT NT NT CFH430 0.047 0.253 0.777CFH396-A NT NT NT CFH461 0.050 0.158 0.957 CFH410-A NA NA NA CFH324-C NANA NA CFH379 NA NA NA CFH338 NA NA NA CFH398-S 0.181 0.327 0.088CFH352-C 14.523  3.818 3.571 CFH412 4.507 2.063 0.869 CFH381-C 0.0520.037 0.105 CFH426 0.263 0.495 0.190 CFH395-C 0.020 0.010 0.010 CFH4401.052 0.534 1.664 CFH409-C 0.086 0.064 0.134 CFH367-S 1.828 2.245 0.692CFH397-2 0.917 0.262 1.662 CFH397-S 1.222 0.315 0.181 CFH381-B 0.0500.060 0.092 CFH383-S 0.937 0.728 0.470 CFH381-M 0.064 0.075 1.761 CFH4110.956 0.384 0.212 CFH395-M 0.050 0.097 0.565 CFH550 NA NA NA CFH421-C0.025 0.063 1.857 CFH384-S 20.638  13.654  NA CFH407 0.086 0.130 1.290CFH412-S 0.087 0.058 3.716 CFH435 0.218 0.161 1.823 CFH426-S 0.117 0.1064.324 CFH449 0.147 0.176 1.784 CFH538 1.898 2.831 NA CFH412-C 6.2742.913 3.068 CFH552 2.600 3.558 NA CFH407-C 0.106 0.052 0.332 CFH325-B0.134 0.188 0.608 CFH449-H 0.011 0.016 0.049 CFH443-5 0.032 0.011 0.488CFH455-C 0.079 0.075 0.075 CFH443-4 0.056 0.106 0.185 ^(a)the testedcompound of 20 μg/mL has no inhibitory activity; ^(b)untested.

It can be seen from the experimental results of the above table: thebiological results of transformation of R₂ part showed that, alkylgroups, the substitution by alkene groups and aryl groups present goodHDAC inhibitory activity. The diversification of the substituent groupis conducive to the biological activity of the compounds.Isobutylene-substituted compound CFH395 has the best inhibitory activityon HDAC 1. Compounds CFH355, CFH437 and CFH417 selectively inhibit HDAC1(HDAC1/HDAC6=˜30 times). Meanwhile compound CFH355 has a 8-timeselectivity to HDAC1 and HDAC3.

At the same time, the biological activity showed that if R₂ partsremains same and Y is —(C₁-C₁₀ alkyl group)-, the length of the chainhas a large impact on the activity. Similarly, if the side chains havethe same length, different R₂-substituted compounds have differentactivity either, such as a series of isobutyl-substituted compounds, thechain with 7 methylenes have the best inhibitory activity on HDAC1 whilefor cyclopropyl-substituted compounds, the chain with 6 methylenes havethe best inhibitory activity.

With R₂ group was isobutyl, inventor has investigated fragment Y of theside chain and different type of Zn-chelating groups (ZBG). As for theinhibition of HDAC1, hydroxamic acid ZBG has the best activity. WhileZBG is α-mercapto ketone, HDAC6 is selectively inhibited, such asCFH398-S.

with R₂ group was cyclopropyl, R_(4b) and R_(5b) substitution wereinvestigated. It can be found from the inhibitory activity of HDAC thatwhen R_(4b) and R_(5b) and C to which they are connected form asix-membered ring, i.e. compound CFH421-C, the inhibitory activity onHDAC1 is the best and HDAC1 is selectively inhibited (HDAC1/HDAC6=75times).

Testing Example 2 Experiment for Testing the Cellular-Level AntitumorActivity

1. Experimental Objective:

The objective is to evaluate the antitumor activity of the compounds bytesting the inhibitory activity of the compounds on the growth of thehuman colon cancer HCT-116 cell lines.

2. Principle of the Test:

The colorimetric assay, MTT assay is for assessing cell viability, whichis based on the metabolic reduction of3-(4,5-dimethyl-2-thiazole)-2,5-diphenyl bromide tetrazolium (MTT).NAD(P)H-dependent cellular oxidoreductase enzymes in the mitochondriamay, under defined conditions, reflect the number of viable cellspresent. These enzymes are capable of reducing the tetrazolium dye MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to itsinsoluble formazan, which has a purple color. After using DMSO todissolve Formazan, microplate reader can be used to measure the opticaldensity at a wavelength of 550/690 nm.

3. Experimental Processing:

Sample processing: The sample was dissolved in DMSO and kept at lowtemperation. DMSO in the final system is limited under a lowconcentration that won't affect the enzyme activity.

Cell viability was detected by using MTT assay. The cells kept in thelogarithmic growth phase are digested by 0.05% trypsin and seededovernight on 96-well plates at 2.0×10³ per well in growth media. Afterseeding, cells are incubated with 5% CO₂ at 37° C. overnight. Sixconcentration gradients of the test compounds are added and plates areincubated at 37 for 72 hr and each concentration of each compound aretriplicated. Then 20 μL of 5 mg/mL MTT are added to each well for 3hours at 37° C., The medium are removed and replaced with 100 μL of DMSOat 37° C. until crystals were dissolved. The absorbance are measuredusing a SpectraMAX 340 microplate reader at 550 nm (L1), with areference wavelength at 690 nm (L2). was plotted against differentconcentrations of the inhibitors. IC₅₀ are obtained by the nonlinearfitting of the (L1-L2) value, which is directly proportional to thenumber of viable cells, against the concentration of the samples.

Data processing and results: The single concentration of the compounds,for example, 20 μg/ml, was used in the preliminary screening and theactivity of the sample was tested. As for the samples that exhibit acertain activity, e.g. the inhibition ratio is greater than 50%, theactivity-dose dependency relationship, IC₅₀/EC₅₀ value, were determined,which could be obtained by the nonlinear fitting of the activity againstthe concentration of the samples. The calculation software for thenonlinear fitting is Graphpad Prism 4, and the model for the fitting issigmoidal dose-response (variable slope). For the mostinhibitor-screening models, the bottom and the top of the fitted curveis set as 0 and 100. Generally, all measurements were duplicated (n≧2),and the results are indicated as mean±SD (Standard Deviation) or SE(standard error). A reported compound doxorubicin (doxorubicin) was usedas positive reference in each measurement.

4. Experimental Results:

4.1: Results of the Compounds on the HCT-116 Human Colon Cancer CellLine:

Sample Cell Viability Sample Cell Viability Sample Cell Viability No.IC₅₀ (μM) No. IC₅₀ (μM) No. IC₅₀ (μM) CFH326 11.143 ± 1.167  CFH381-C0.438 ± 0.018 CFH403 7.596 ± 0.817 CFH369 8.714 ± 1.227 CFH395-C 0.408 ±0.023 CFH421-C 0.233 ± 0.029 CFH383 7.406 ± 1.473 CFH409-C 2.976 ± 0.188CFH407 0.310 ± 0.032 CFH382 10.304 ± 1.130  CFH381-B 0.562 ± 0.071CFH435 0.768 ± 0.099 CFH410 6.696 ± 0.652 CFH381-M 0.472 ± 0.055 CFH4490.392 ± 0.031 CFH367-C 0.452 ± 0.022 CFH395-M 0.538 ± 0.051 CFH401 0.267± 0.007 CFH461 7.647 ± 1.499 CFH407-C 1.117 ± 0.484 CFH396 8.266 ± 0.858CFH409-A 15.125 ± 2.919  CFH508 9.322 ± 0.433 CFH421 10.023 ± 0.748 

4.2: Results of the Compounds on the BxPC-3 Human Colon Cancer CellLine:

Cell Via- bility Cell Cell Sample IC₅₀ Sample Viability Sample ViabilityNo. (μM) No. IC₅₀ (μM) No. IC₅₀ (μM) CFH382 2.6 CFH355 3.3 CFH461 0.9CFH396 4.3 CFH410 4.7 CFH383 2.6 CFH395 1.9 CFH424 17.8 CFH403 3.9CFH367 6.2 CFH326 6.8 CFH367-C 1.3 CFH397-2 3.7 CFH340-M 13.0 CFH417 1.9CFH352 2.1 CFH369 4.8 CFH397 6.4 CFH421 4.8

4.3: Results of the Compounds on Other Tumor Cell Lines:

HL60 A549 MDA-MB-231 HMEC Sample No. IC₅₀ (μM) IC₅₀ (μM) IC₅₀ (μM) IC₅₀(μM) CFH395 0.3 1.6 0.5 1.2 CFH367-C 0.5 2.8 0.7 1.7 CFH383-4 5.1 32.09.4 22.0 CFH382 NT^(a) 3.6 2.7 NT CFH417 NT 3.2 2.1 NT ^(a)untested.

5. Conclusions: the compounds having inhibitory activity on HDAC1without exception exhibit good activity in inhibiting tumor cellproliferation, in particular has a good impact on colon cancer,pancreatic cancer, adenocarcinoma of the lung, breast cancer. From thepoint of the inhibitory growth activity of the cancer cell lines, theactivity of the compound on the cell is substantially consistent withthat on the enzymes.

Testing Example 3 HDAC Inhibitor as the Drug for Treating MultipleSclerosis (MS)

1. Experimental Method and Results:

The Clinical Score of EAE can be Effectively Alleviated by HDACiCFH367-C.

The inventor carried out a hypodermic injection of 100 μL MOG₃₅₋₅₅emulsified by Freund's complete adjuvant (150 μg/mice) and MT toxin (5mg/mL) by thermal inactivation on the 8-week-old female C57 mice. And,on the same day and on the third day intraperitoneal injection of PTX(200 ng/mice/time, dissolved by PBS) is carried out twice. On the thirdday after immunization, the mice are administered via gavage, twicedaily, and each dose is 10 mg/kg of the body weight. The performance ofthe mice were observed daily and rated based on the following criteria:0: no signs of incidence; tail with weakness (0.5 points) or paralysis(1 point); all fours with weakness (0.5 points) or paralysis (1 point).The above scores are counted up to achieve a final score. The resultsshow that (FIG. 1), CFH367-C has a good therapeutic effect on theclinical symptoms of EAE, and the severity of the disease in the treatedmice was significantly lower than control group of the solvent (P<0.01).

HDACi CFH367-C Significantly Reduced the Demyelination Phenomenon ofSpinal Cords of EAE Animal.

The samples of spinal cords from normal control, EAE mice and thetreatment group of EAE mice were taken respectively. After being fixed,the samples were envoloped by conventional paraffin embedding, sectionin 5 micrometers, hydration with dewaxing to 95% alcohol, staining withLFB (Luxol Fast Blue) overnight at 56 degree; washing by 95% ethanol andthen by water, color separation using a 0.05% lithium carbonatesolution, washing by 70% ethanol twice, washing by distilled water,staining with eosin for 2 minutes, gradient ethanol dehydration,transparency treatment by xylene, and mounting by neutral gum. The maincoloring parts of LFB is the white matter of the spinal cord, mainlycomposed of nerve fibers and their myelin. Experimental results showedthat the white matter of normal mice can be dyed blue with a relativelydense structure; while the white matter regions of spinal cord in EAEmice appeared large number of vacuoles, and the degree of coloration wassignificantly reduced, showing a severe demyelination phenomenon. Themice administered with CFH367-C had a dense structure of the whitematter of the spinal cord with an even coloring. This was close to thenormal control mice, showing its significant therapeutic effect.

HDACi CFH367-C Significantly Reduced the Infiltration of the PeripheralImmune Cells in the Spinal Cord of EAE Animal.

The samples of spinal cords from normal control, EAE mice and thetreatment group of EAE mice were taken respectively. After being fixed,the samples were treated by the following steps in sequence, which wereconventional paraffin embedding and section in 5 micrometers. Thesections were dewaxed by xylene, rehydrated by all levels of ethanol,and dyed with hematoxylin for five minutes; After that the slices werewashed by running water, and the color was separated by hydrochloricacid. Then after water washing, the slices were dyed with eosin for 2minutes; after dehydration by gradient ethanol dehydration, the sliceswere made transparent by xylene, and mounted with neutral gum. Theexperimental results show that almost no peripheral immune cells wasinfiltrated in the spinal cord of the normal mice; while the whitematter regions in spinal cord of EAE mouse appeared a large number ofperipheral immune cell with infiltration, and this caused the damage ofannex tissue with the appearance of the vacuoles. The white matterstructure of the spinal cord in the mice administered with CFH367-C isdense with no obvious infiltration in the peripheral immune cells,showing a significant therapeutic effect.

HDACi CFH367-C Significantly Reduced the Infiltration of CD54 PositiveLeukocyte in the Spinal Cord of EAE Animal.

The HDACi CFH367-C significantly reduced EAE animal spinal cordCD45-positive leukocyte infiltration. Spinal cords is taken from themice. After being fixed, the spinal cords are treated with OCT and madeto be 10 μm frozen sections. The sections were washed with PBS threetimes, and each time for 5 min, then incubated overnight at 4° C. withantibody against CD45 (primary antibody); after being washed with PBSthree times, the sections are dyed with fluorescence-labeled secondaryantibody at 37° C. for 1 h. After being washed with PBS three times, thesections are mounted with glycerine. CD45 is the common antigen ofleukocyte. The experimental results showed that nuclear staining showedthat there are a large number of cells aggregated in the spinal cord ofEAE mice, and most of them were the CD45 positive leukocytes. Thisphenomenon did not exist in normal mice, while the aggregation ofpositive leukocyte also has not been observed in the mice which wereadministered with CFH367C, showing that the infiltration of theleukocyte to the spinal cord of EAE mice can be inhibited by CFH367-C.

HDACi CFH367-C Significantly Reduced the Infiltration of CD4 Positive TCells in the Spinal Cord of EAE Animal.

Preliminary studies have indicated that CD4 positive T cells play animportant role in the pathopoiesis of EAE. The inventor has alsoobserved the function of the drugs on the filtration of CD4 positive Tcells. Spinal cords were taken from the mice. After being fixed, thespinal cords were embedded in OCT to be made into 10 μm frozen sections.The sections were washed with PBS three times, and each time for 5 min,then incubated overnight at 4° C. with antibody (primary antibody)against CD4; After being washed with PBS three times, the sections aredyed with fluorescence-labeled secondary antibody at 37° C. for 1 h.After being washed with PBS three times, the sections are mounted byglycerine. The experimental results showed that nuclear staining showedthat there are a large number of cells aggregated in the spinal cord ofEAE mice, and most of them were the CD4 positive T cells. Thisphenomenon did not exist in normal mice. The filtration and aggregationof the CD4 positive T cells can be obviously alleviated by administeringCFH367-C.

2. Conclusions:

Histone deacetylase inhibitor CFH367-C can effectively alleviate theclinical symptoms of experimental mouse model EAE of MS. It is found bydyeing the spinal cord of EAE mice that CFH367-C can inhibit theperipheral immune cells, especially the infiltration of CD4 positive Tcell to the central nervous system of the mice, and alleviatedemyelinating phenomenon of the neurons of EAE animal and furtheralleviate the clinical manifestations of EAE. The present researchresults indicated that histone deacetylase inhibitor CFH367-C can beused in the treatment of MS disease and may be applied into treatmentsof other autoimmune diseases, including rheumatoid arthritis, psoriasis,systemic lupus erythematosus etc.

1. A thiazole compound of general formula I,

wherein, R₁ is R_(1a), R_(1b) or R_(1c):

In which, R_(4a) and R_(5a) each independently are A group or C₁-C₆alkyl optionally substituted by tert-butoxycarbonylamino; R_(4b) andR_(5b) each independently are A group; or, R_(4b) and R_(5b) togetherwith the carbon atom to which they are attached form a 3 to 10-memberedcyclic hydrocarbon or a 3 to 10-membered heterocycle containing 1 to 3heteroatoms selected from N, O and S; R_(4c), R_(5c) and R₆ eachindependently are A group; The said A group is hydrogen, halogen,hydroxyl, nitro, C₃-C₆ cycloalkyl, C₁-C₆ alkoxyl, C₁-C₆ alkoxyloptionally substituted by C₆-C₁₀ aryl, amino, C₁-C₆ alkylamino, C₁-C₆alkylamino optionally substituted by C₆-C₁₀ aryl, C₁-C₆ alkyl, C₁-C₆alkyl optionally substituted by hydroxyl, C₁-C₆ alkyl optionallysubstituted by C₁-C₄ alkoxyl, C₁-C₆ alkyl optionally substituted byfluoro, C₁-C₆ alkyl optionally substituted by C₆-C₁₀ aryl, C₂-C₆alkenyl, C₂-C₆ alkenyl optionally substituted by hydroxyl, C₂-C₆ alkenyloptionally substituted by C₁-C₄ alkoxyl, C₂-C₆ alkenyl optionallysubstituted by fluoro, C₂-C₆ alkenyl group optionally substituted byC₆-C₁₀ aryl, C₂-C₆ alkynyl, C₂-C₆ alkynyl optionally substituted byhydroxyl, C₂-C₆ alkynyl optionally substituted by C₁-C₄ alkoxyl, C₂-C₆alkynyl optionally substituted by fluoro, C₂-C₆ alkynyl optionallysubstituted by C₆-C₁₀ aryl, C₆-C₁₀ aryl, or 5 to 7-membered aromaticheterocycle containing 1-3 heteroatoms selected from N, O and S; R₂ ishydrogen, halogen, hydroxyl, C₁-C₆ alkoxyl, C₁-C₈ alkoxyl optionallysubstituted by C₆-C₁₀ aryl, amino, C₁-C₆ alkylamino, C₁-C₆ alkylaminooptionally substituted by C₆-C₁₀ aryl, C₁-C₆ alkyl, C₃-C₆ cycloalkyl,C₃-C₆ cycloalkyl optionally substituted by C₁-C₆ alkyl, C₁-C₆ alkyloptionally substituted by one or more substituent groups independentlyselected from hydroxyl, C₁-C₄ alkoxyl, halogen, benzyloxyl and C₆-C₁₀aryl, C₂-C₈ alkenyl, C₂-C₆ alkenyl group optionally substituted by oneor more substituent groups independently selected from hydroxyl, C₁-C₄alkoxyl, halogen and C₆-C₁₀ aryl, C₂-C₆ alkynyl, C₂-C₈ alkynyloptionally substituted by one or more substituent groups independentlyselected from hydroxyl, C₁-C₄ alkoxyl, halogen and C₆-C₁₀ aryl, C₆-C₁₀aryl, C₆-C₁₀ aryl optionally substituted by halogen or nitro, or 5-7membered aromatic heterocycle containing 1 to 3 heteroatoms selectedfrom N, O and S; n is 0, 1 or 2; X is —N(R₇)— or

wherein R₇ is hydrogen or C₁-C₆ alkyl; Y may do not exist or exist as—(C₁-C₁₀ alkyl)-, —(C₂-C₉ alkenyl)-, —(C₆-C₁₀ aryl)-, —(C₁-C₆alkyl)-(C₆-C₁₀ aryl)-, —(C₆-C₁₀ aryl)-(C₂-C₆ alkenyl)-, —(C₃-C₆cycloalkyl)-, —(C₁-C₅ alkyl)-C(O)—NH—(C₁-C₅ alkyl)-, —(C₁-C₅alkyl)-C(O)—O—(C₁-C₅ alkyl)- or —(C₁-C₅ alkyl)-C(O)—O—(C₂-C₉ alkenyl)-;R₃ is R_(3a), R_(3b), R_(3c), R_(3d) or R_(3e):

R₈ is hydrogen or C₁-C₆ alkylcarbonyl; R₉ is hydrogen or C₁-C₁₀alkylcarbonyl.
 2. The compound according to claim 1, wherein, In thegeneral formula I: R₂ is hydrogen, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, C₁-C₆alkyl optionally substituted by hydroxyl, benzyloxyl or C₆-C₁₀ aryl,C₂-C₈ alkenyl, C₆-C₁₀ aryl, or C₆-C₁₀ aryl optionally substituted byhalogen or nitro; n is 0; X is —NH—; Y is —(C₁-C₁₀ alkyl)-, —(C₆-C₁₀aryl)-, —(C₁-C₆ alkyl)-(C₆-C₁₀ aryl)- or —(C₆-C₁₀ aryl)-(C₂-C₆alkenyl)-; R₈ is hydrogen.
 3. The compound according to claim 1, whereinthe compound has the structure of general formula II_(a), II_(b) orII_(c):

In the general formula II_(a): R₂, X, Y and R₃ are defined as stated inclaim 1; R_(4a) and R_(5a) each independently are hydrogen, C₁-C₆ alkylor C₁-C₆ alkyl optionally substituted by tert-butoxycarbonylamino; Inthe formula II b: R₂, X, Y and R₃ are defined as stated in claim 1;R_(4b) and R_(5b) each independently are hydrogen, C₁-C₆ alkyl, C₃-C₆cycloalkyl or C₆-C₁₀ aryl; or, R_(4b) and R_(5b) together with thecarbon atom to which they are attached form a 3 to 10-membered cyclichydrocarbon; In the formula II_(c): R₂, X, Y and R₃ are defined asstated in claim 1; R_(4c), R_(5c) and R₆ each independently are hydrogenor C₁-C₆ alkyl.
 4. The compound according to claim 1, wherein thecompound has the structure of general formula IV:

In the formula IV, R₂, R_(4b), R_(5b) and Y each are defined as statedin claim
 1. 5. The compound according to claim 4, wherein in generalformula IV: R₂ is hydrogen, C₁-C₆ alkyl, C₃-C₆ cycloalkyl, C₁-C₆ alkyloptionally substituted by hydroxyl, C₃-C₆ cycloalkyl optionallysubstituted by C₁-C₆ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkenyl optionallysubstituted by hydroxyl, C₆-C₁₀ aryl, C₁-C₆ alkyl optionally substitutedby C₆-C₁₀ aryl or benzyloxyl, C₂-C₈ alkenyl optionally substituted byC₆-C₁₀ aryl, C₂-C₈ alkoxyl optional substituted by C₆-C₁₀ aryl, C₆-C₁₀aryl optionally substituted by halogen, or C₆-C₁₀ aryl optionallysubstituted by nitro; R_(4b) and R_(5b) each independently are hydrogen,fluoro, C₁-C₆ alkyl, C₁-C₆ alkyl optionally substituted by hydroxyl,C₁-C₆ alkyl optionally substituted by fluoro, C₃-C₆ cycloalkyl, C₂-C₆alkenyl, C₂-C₆ alkenyl optionally substituted by hydroxyl, C₂-C₆ alkenyloptionally substituted by fluoro, or C₆-C₁₀ aryl; or, R_(4b) and R_(5b)together with the carbon atom to which they are attached form a 3 to10-membered cyclic hydrocarbon or a 3 to 10-membered heterocyclecontaining 1 to 3 heteroatoms selected from N, O and S; Y is —(C₁-C₈alkyl)-, —(C₆-C₁₀ aryl)-, —(C₁-C₆ alkyl)-(C₆-C₁₀ aryl), or —(C₆-C₁₀aryl)-(C₂-C₆ alkenyl)-.
 6. The compound according to claim 4, wherein,the compound is selected from the group consisting of:


7. A method for preparing the compound according to claim 1, wherein,the method can be employed by any one of the following Route one toRoute five;

In Route one: R₁, R₂, n, X and Y each have the same meaning as stated inclaim 1; Compound 1 reacts with active zinc powder in THF to form zincreagent compound 2; Negishi coupling reaction of compound 2 with2-bromo-thiazole compound catalyzed by palladium acetate andtriphenylphosphine in toluene affords compound
 4. Hydrolysis of compound4 in sodium hydroxide/methanol-water produces compound 5 which is thecorresponding acid of compound 4; Condensation reaction of compound 5with various nucleophilic reagents of the formula HX—Y—COOMe isconducted using condensing agents such as EDCI, HOBt and i-Pr₂NEt in DMFto obtain compound 6; Compound 6 reacts with the freshly-made methanolsolution of hydroxy amine to produce said compound I_(a);

In Route two: R₁, R₂, R₈, n, X and Y each have the same meaning asstated in claim 1; Compound 6 is hydrolyzed by alkali in methanol orTHF/water to obtain Compound I_(e); Condensation reaction of compoundI_(e) with mono-Boc-protected o-phenylenediamine is conducted usingcondensing agents such as EDCI, HOBt and i-Pr₂NEt in DMF to obtaincompound 7; Boc protecting group of compound 7 is removed inhydrochloric acid ethyl acetate solution to obtain said compounds I_(b),or compound 7 reacts with various nucleophile of formula R₈H to obtainsaid compound I_(b);

In Route three: R₁, R₂, n, X and Y each have the same meaning as statedin claim 1; Condensation reaction of compound 5 with various nucleophileof the formula HX—Y—NHCOCH₂STrt is conducted using condensing agentssuch as EDCI and DMAP as alkali in dichloromethane to obtain compound 8;Boc protecting group of compound 8 is removed by the trifluoroaceticacid to obtain said compound I_(c);

In Route four: R₁, R₂, R₉, n, X and Y each have the same meaning asstated in claim 1; Condensation reaction of compound 5 with variousnucleophile of the formula HX—Y—SR₉ is conducted using condensing agentssuch as EDCI, HOBt and i-Pr₂NEt in DMF to obtain said compound I_(d);

In Route five: R₂, R₃, R_(4c), R_(5c), R₆, n, X and Y each have the samemeaning as stated in claim 1; Reaction of compound 9 with Lawesson'sagent affords compound 10; Hantzsch reaction of compound 10 with ethylbromopyruvate substituted by R₂ produces bisthiazole compound 11;Compound 11 is hydrolyzed by alkaline in methanol/water to affordcompound 12; Condensation reaction of compound 12 with variousnucleophile of the formula HX—Y—R₃ is conducted using condensing agentssuch as EDCI, HOBt and i-Pr₂NEt in DMF to obtain said compound II_(c).8. A use of the compound according to claim 1 for preparingpharmaceuticals as histone deacetylase inhibitors.
 9. A use of thecompound according to claim 1 for preparing pharmaceuticals againsttumor.
 10. The use according to claim 9, wherein, said tumor is selectedfrom colon cancer, pancreatic tumor, leukemia, lung tumor or breasttumor.
 11. A use of the compound according to claim 1 for preparingpharmaceuticals for the treatment of multiple sclerosis.